S protein mutants of novel coronavirus variant strains and genetically engineered mrna and vaccine compositions thereof

Abstract

The present application mutates multiple amino acid residues to proline in the C-terminal amino acids of the ectodomain of the spike protein (S protein) of the novel coronavirus variant strain, provides an S protein mutant with stable conformation trimer, and constructs an mRNA encoding the above-mentioned S protein mutant, and a vector capable of transcribing the mRNA in vitro to prepare the mRNA. The S protein mutant, and the mRNA (including further optimized mRNA) encoding it, can be used to induce an immune response against the novel coronavirus in a subject, thereby preventing and / or treating diseases or conditions associated with novel coronavirus infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and vaccine technology, and particularly relates to recombinant antigens for preparing vaccines against variant strains of the novel coronavirus (2019-nCoV), their genetically engineered mRNAs and vectors, and their mRNA vaccine compositions. Background Technology

[0002] Traditional inactivated vaccines, live attenuated vaccines, and peptide vaccines have long development cycles and complex manufacturing processes. However, mRNA vaccines, based on advancements in mRNA modification and delivery tools, can be rapidly designed and manufactured on a clinical scale within weeks once the viral antigen sequence is obtained. This allows for standardized production, making them highly attractive in responding to pandemic outbreaks of infectious diseases. Furthermore, mRNA vaccines do not carry the potential reversal risk of live attenuated vaccines or the reversion mutation problem of inactivated vaccines. In terms of immunogenicity, mRNA vaccines can induce B-cell and T-cell immune responses, generate immune memory, and express multiple antigens simultaneously, delivering more effective antigens. Moreover, mRNA only needs to cross the cell membrane to efficiently express antigen proteins in the cytoplasm, without the risk of gene integration into the genome. Finally, mRNA is easily degraded after translation into protein, and its transient expression characteristic not only ensures the safety of mRNA drugs but also allows for dosage control, avoiding antigen tolerance caused by long-term exposure to vaccine drugs. Therefore, mRNA vaccines have disruptive advantages in terms of safety, rapid preparation, and immunogenicity.

[0003] mRNA is transcribed from a DNA template strand, and its sequence is identical to the coding strand and complementary to the template strand. Unlike prokaryotes, in eukaryotes, the mRNA carrying genetic information consists of protein-coding exons and non-coding introns arranged in intervals. Only properly modified and spliced ​​mature mRNA can serve as a template for transport to the cytoplasm for further translation into proteins.

[0004] The novel coronavirus (2019-nCoV, also known as SARS-CoV-2) is spherical or ellipsoidal in shape, with a diameter of 80-120 nm. Under an electron microscope, the surface of the virus particle has ball-and-bar shaped protrusions composed of the trimeric spike glycoprotein (S). The viral envelope is composed of membrane glycoprotein (M), which is embedded in the viral envelope through three transmembrane domains. In addition, a small amount of small transmembrane protein—the envelope (E) protein—is also present in the envelope. Finally, the nucleocapsid (N) protein is attached to the RNA genome in a beaded manner, forming a helical symmetrical nucleocapsid. The results show that S, M, E, and N proteins are the main components of the immune response induced by coronaviruses. Furthermore, the receptor-binding domain (RBD) in the S protein infects human respiratory epithelial cells by interacting with the human ACE2 protein.

[0005] The 2019-nCoV mutation occurs frequently. After circulating in the population for a period of time, certain dominant mutant strains will emerge. By leveraging the advantages of mRNA vaccine development, designing and producing mRNA vaccines targeting these dominant mutant strains can help address the immune escape from newly emerging mutant strains. Summary of the Invention

[0006] This invention involves mutating multiple amino acid residues to proline in the C-terminal amino acid domain of the spike protein (S protein) of a 2019-nCoV mutant strain, providing an S protein mutant with a stable conformational trimer. The invention also includes the construction of mRNA encoding the aforementioned S protein mutant and a vector capable of in vitro transcription to prepare the mRNA. These S protein mutants, and their encoding mRNAs (including further optimized mRNAs), can be used to induce an immune response in subjects against both wild-type and mutant 2019-nCoV strains, thereby preventing and / or treating diseases or conditions associated with 2019-nCoV infection.

[0007] The main known structures of the 2019-nCoV virus particle include single-stranded positive-sense nucleic acid, spike protein (S), membrane protein (M), envelope protein (E), and nucleocapsid protein (N). For example... Figure 1As shown, the S protein can be divided into the receptor-binding subunit S1 and the membrane fusion subunit S2. The adsorption and invasion of cells by the 2019-nCoV virus mainly relies on the S protein. In this process, the S protein assembles in the form of a homotrimer, with its cytoplasmic tail and transmembrane domain anchoring it to the viral membrane. Analysis of the pre-fusion structure of the S protein revealed that the RBD of the S1 subunit undergoes hinge-like conformational shifts to hide or expose key receptor-binding sites. "Down" represents the receptor-unbinding state, and "up" represents the receptor-binding state, indicating a relatively unstable state. This conformation allows the S protein to easily bind to the host receptor angiotensin-converting enzyme 2 (ACE2). After the RBD binds to the receptor, the S2 subunit changes to the post-fusion conformation by inserting its FP into the host cell membrane. Cryo-electron microscopy experiments identified numerous trimeric S protein domains in the pre-fusion conformation, and a large number of neutralizing antibody-sensitive epitopes were present on the pre-fusion S protein. The post-fusion conformation minimizes the exposure of these neutralizing antibody-sensitive epitopes, which are only present in the pre-fusion conformation.

[0008] Therefore, if it is to be used as an antigen in a vaccine, the optimized S protein mutant should be able to retain the epitope present in the prefusion conformation of the S protein and induce antibodies that can inhibit viral fusion.

[0009] S protein mutants arise from amino acid mutations in the parental S protein, including substitutions, deletions, and / or insertions. The parental S protein can be the S protein of the 2019-nCoV wild-type strain or the S protein of any mutant strain of 2019-nCoV (the mutation in any mutant strain of 2019-nCoV can occur in the S protein region or in a non-S protein region). The parental S protein can be the full-length S protein or a fragment of the full-length S protein (e.g., a truncated sequence compared to the full-length S protein (e.g., deletion of the cytoplasmic tail and / or transmembrane domain)).

[0010] In this invention, the amino acid positions of the S protein mutant and the parental S protein are described based on the amino acid sequence of the wild-type S protein. The amino acid sequence of the wild-type S protein can be obtained from NCBI GeneID:43740568 and contains 1273 amino acids. Its sequence is shown below and is marked as SEQ ID NO:1 in this invention.

[0011] In one embodiment of the present invention, the parental S protein is the S protein of the 2019-nCoV B.1.617.2 mutant strain, which has the following mutations compared to the S protein of the 2019-nCoV wild strain: T19R, G142D, EF156_157del, R158G, L452R, T478K, D614G, P681R, D950N (the sites are described by their positions in the amino acid sequence shown in SEQ ID NO:1).

[0012] The first aspect of the present invention is to provide an S protein mutant.

[0013] According to the present invention, the S protein mutant includes at least an extracellular domain, the extracellular domain of which, relative to the extracellular domain of the parental S protein, comprises amino acid mutations at the following positions: F817P, A892P, A899P, A942P, and KV986_987PP, the sites being located and described by the amino acid sequence shown in SEQ ID NO:1. These amino acid mutations can improve the stability of the S protein mutant.

[0014] In some embodiments of the present invention, the S protein mutant also has the following mutations relative to the parental S protein: T19R, G142D, EF156_157del, R158G, L452R, T478K, D614G, P681R, D950N, the sites being located and described by the amino acid sequence shown in SEQ ID NO:1.

[0015] According to the present invention, in some embodiments, the S protein mutant has a mutation at the furinase cleavage site relative to the parental S protein, specifically by mutating amino acids 682-685 (described by the position of the amino acid sequence shown in SEQ ID NO:1) RRAR to lose its ability to be cleaved by furin-like proteases. In one embodiment of the present invention, RRAR is mutated to GSAS. By mutating the cleavage site in the S protein, the S protein mutant can be prevented from being cleaved by proteases, further improving its stability.

[0016] According to some embodiments of the present invention, the S protein mutant does not contain the transmembrane domain and / or cytoplasmic tail of the S protein.

[0017] According to the present invention, in some embodiments, the S protein mutant may also have an amino acid mutation in the fusion peptide domain relative to the parental S protein. By substituting, deleting, and / or inserting one or more amino acid residues in this region, the fusion peptide domain loses its native function, i.e., its function of mediating viral fusion with the host cell membrane. In some embodiments, the S protein mutant does not contain a fusion peptide domain. By inducing a mutation in the fusion peptide domain in the S protein mutant to render it nonfunctional, the stability of the pre-fusion conformation of the S protein mutant can be improved, thereby preserving and exposing the large number of neutralizing antibody-sensitive epitopes present on the pre-fusion conformation of the S protein.

[0018] According to the present invention, in some embodiments, the S protein mutant is directly fused with a trimer-forming domain at the C-terminus of its extracellular region (amino acids 1-1209, the site being described by the position of the amino acid sequence shown in SEQ ID NO:1). A “trimer-forming domain” refers to a protein or polypeptide domain that, when expressed, can spontaneously or inducedly form a trimer. Various such domains are known in the art. By including a trimer-forming domain in the S protein mutant (e.g., by constructing a fusion protein), it is possible to promote the formation of a trimer conformation of the S protein mutant and / or stabilize the trimer conformation of the S protein mutant. In one embodiment of the present invention, the trimer-forming domain is a T4 Fibritin Foldon Trimerization Motif. In a specific embodiment of the present invention, the amino acid sequence of the T4 Fibritin Foldon Trimerization Motif is shown in SEQ ID NO:3.

[0019] In some preferred embodiments of the present invention, the S protein mutant of the present invention is described by the amino acid sequence shown in SEQ ID NO:1, having six proline mutations in the extracellular domain of the S protein: F817P, A892P, A899P, A942P, and KV986_987PP; and the following mutations: T19R, G142D, EF156_157del, R158G, L452R, T478K, D614G, P681R, D950N; and a mutation of amino acids 682-685 from RRAR to GSAS; and lacking the transmembrane domain and cytoplasmic tail of the S protein. In one embodiment of the present invention, the S protein mutant comprises the amino acid sequence shown in SEQ ID NO:2.

[0020] In some preferred embodiments of the present invention, the S protein mutant of the present invention is described by the position of the amino acid sequence shown in SEQ ID NO:1. The mutant has six proline mutations in the extracellular domain of the S protein: F817P, A892P, A899P, A942P, and KV986_987PP; and the following mutations: T19R, G142D, EF156_157del, R158G, L452R, T478K, D614G, P681R, D950N; and a mutation of amino acids 682-685 from RRAR to GSAS; and does not contain the transmembrane domain and cytoplasmic tail of the S protein; and directly fuses the T4 Fibritin FoldonTrimerization Motif, a domain that helps form a trimer, to the C-terminus of the extracellular region. In one embodiment of the present invention, the S protein mutant comprises the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:3 directly linked from the N-terminus to the C-terminus. In one embodiment of the present invention, the amino acid sequence of the S protein mutant is the amino acid sequence of SEQ ID NO:2 and the amino acid sequence of SEQ ID NO:3 directly linked from the N-terminus to the C-terminus.

[0021] A second aspect of the present invention is to provide a DNA molecule, an expression vector or cell containing said DNA molecule, said DNA molecule being used to encode the S protein mutant described in the first aspect of the present invention.

[0022] According to the present invention, the DNA molecule may be present in an expression vector, such as a plasmid vector or a viral vector, and transfected into engineered cells for expression to obtain the S protein mutant of the present invention. Alternatively, the DNA molecule may be recombined into the genome of engineered cells and expressed in the engineered cells to obtain the S protein mutant of the present invention.

[0023] In some embodiments of the present invention, the nucleotide sequence of the DNA molecule comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% homologous to the nucleotide sequence shown in SEQ ID NO:4, wherein the nucleotide sequence shown in SEQ ID NO:4 encodes the amino acid sequence shown in SEQ ID NO:2.

[0024] In some embodiments of the present invention, the nucleotide sequence of the DNA molecule comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% homologous to the nucleotide sequence shown in SEQ ID NO:5, wherein the nucleotide sequence shown in SEQ ID NO:5 encodes the amino acid sequence shown in SEQ ID NO:3.

[0025] In some embodiments of the present invention, the nucleotide sequence of the DNA molecule comprises a nucleotide sequence directly linked from the 5' end to the 3' end that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% homologous to the nucleotide sequence of SEQ ID NO:4, and a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% homologous to the nucleotide sequence of SEQ ID NO:5. In one specific embodiment of the present invention, the nucleotide sequence of the DNA molecule comprises the nucleotide sequence of SEQ ID NO:4 and the nucleotide sequence of SEQ ID NO:5 directly linked from the 5' end to the 3' end.

[0026] An expression vector containing the DNA molecule. According to the present invention, the expression vector may be a prokaryotic cell expression vector or a eukaryotic cell expression vector.

[0027] A cell containing the DNA molecule. According to the invention, the DNA molecule may be present outside the genome of the cell or may be recombined into the genome of the cell.

[0028] A third aspect of the present invention is to provide an mRNA that encodes the S protein mutant described in the first aspect of the present invention.

[0029] According to the present invention, the mRNA contains an open reading frame (ORF) encoding a mutant S protein.

[0030] According to the present invention, the mRNA may include a 5' cap structure, a 5' UTR, an open reading frame (ORF) encoding the S protein mutant, a 3' UTR, and a poly-A tail from the 5' end to the 3' end.

[0031] 5' Cap Structure: A 5' cap is typically a modified nucleotide (especially a guanine nucleotide) added to the 5' end of an mRNA molecule, but atypical cap analogs are also included. Preferably, the 5' cap is added using a 5'-5'-triphosphate bond (also known as m7GpppN). In some embodiments of the invention, the 5' cap structure is CAP1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), CAP2 (additional methylation of the ribose of the second nucleotide downstream of m7GpppN), CAP3 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), and CAP4 (additional methylation of the ribose of the fourth nucleotide downstream of m7GpppN).

[0032] The 5' cap structure can be formed using cap analogs in chemical RNA synthesis or in vitro RNA transcription (co-transcriptional capping), or in vitro using capping enzymes (e.g., commercially available capping kits).

[0033] In one embodiment of the present invention, the 5' cap structure is a Cap1 structure.

[0034] According to the present invention, the 5'UTR may comprise the 5'UTR of β-globin or α-globin, or its homologs or fragments. In some embodiments of the present invention, the 5'UTR comprises the 5'UTR of β-globin, or its homologs or fragments. In some embodiments of the present invention, the 5'UTR comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% homologous to the 5'UTR nucleotide sequence of β-globin shown in SEQ ID NO:6. In one specific embodiment of the present invention, the 5'UTR comprises the 5'UTR nucleotide sequence of β-globin shown in SEQ ID NO:6.

[0035] In some embodiments of the present invention, the 5'UTR further comprises a Kozak sequence. In one embodiment of the present invention, the Kozak sequence is GCCACC.

[0036] According to the present invention, the 3'UTR may comprise the 3'UTR of β-globin or α-globin or its homologs, fragments, or combinations of fragments. In some embodiments of the present invention, the 3'UTR comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% homologous to the fragment of the α2-globin 3'UTR shown in SEQ ID NO:7. In other embodiments of the present invention, the 3'UTR comprises two end-to-end nucleotide sequences that are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% homologous to the fragment of the α2-globin 3'UTR shown in SEQ ID NO:7. In one specific embodiment of the present invention, the 3'UTR comprises two end-to-end nucleotide sequences shown in SEQ ID NO:7.

[0037] According to the present invention, the length of the poly-A tail can be 50-200 nucleotides, preferably 100-150 nucleotides, for example 110-120 nucleotides, for example about 110 nucleotides, about 120 nucleotides, about 130 nucleotides, about 140 nucleotides, or about 150 nucleotides.

[0038] In one embodiment of the present invention, the nucleotide sequence of the open reading frame (ORF) of the S protein mutant is a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% homologous to the nucleotide sequence shown in SEQ ID NO:8. The amino acid sequence of the S protein mutant translated from the ORF consists of the amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:3 directly linked from the N-terminus to the C-terminus. In a specific embodiment of the present invention, the nucleotide sequence of the open reading frame (ORF) of the S protein mutant is as shown in SEQ ID NO:8.

[0039] In one embodiment of the invention, the mRNA comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% homologous to the nucleotide sequence shown in SEQ ID NO:9. In a specific embodiment of the invention, the mRNA comprises the nucleotide sequence shown in SEQ ID NO:9.

[0040] According to the present invention, one or more nucleotides in the mRNA may be modified. For example, one or more nucleotides (e.g., all nucleotides) in the mRNA may each be independently replaced with naturally occurring or artificially synthesized nucleotide analogs.

[0041] A fourth aspect of the present invention is to provide a nucleic acid molecule encoding the mRNA described in the third aspect of the present invention. The nucleic acid molecule may be, for example, in the form of a vector such as a plasmid vector or a viral vector. In some embodiments, the nucleic acid molecule may be used to prepare the mRNA of the present invention in vitro by transcription.

[0042] In one embodiment of the invention, the nucleic acid molecule is an in vitro transcription vector comprising operatively linked nucleotide sequences encoding a 5'UTR, a 3'UTR, and a poly-A tail. The 5'UTR comprises nucleotide sequences with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% homology to the 5'UTR nucleotide sequence of β-globin shown in SEQ ID NO:6. The 3'UTR comprises two end-to-end nucleotide sequences with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% homology to the 3'UTR of α2-globin shown in SEQ ID NO:7. The length of the poly-A tail can be 50-200 nucleotides, preferably 100-150 nucleotides, such as 110-120 nucleotides, such as about 110 nucleotides, about 120 nucleotides, about 130 nucleotides, about 140 nucleotides, or about 150 nucleotides.

[0043] According to the present invention, the in vitro transcription vector further comprises a nucleotide sequence encoding an open reading frame (ORF) of an S protein mutant. The nucleotide sequence of the open reading frame (ORF) of the S protein mutant is a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% homologous to the nucleotide sequence shown in SEQ ID NO:8.

[0044] In one specific embodiment of the present invention, the in vitro transcription vector comprises operatively linked nucleotide sequences encoding a 5'UTR, an ORF of the S protein mutant, a 3'UTR, and a poly-A tail; the 5'UTR comprises the 5'UTR nucleotide sequence of β-globin as shown in SEQ ID NO:6; the 3'UTR comprises two end-to-end nucleotide sequences as shown in SEQ ID NO:7; the poly-A tail is 50-200 nucleotides in length; and the nucleotide sequence of the open reading frame (ORF) of the S protein mutant is shown in SEQ ID NO:8.

[0045] According to the present invention, commonly used plasmids can be used as vectors. In some embodiments of the present invention, the plasmid is psp73 or pUC57-kana.

[0046] The mRNA of this invention can be prepared using methods known in the art, including but not limited to chemical synthesis or in vitro transcription. In some embodiments of this invention, nucleic acid molecules encoding mRNA can be artificially synthesized, cloned into a vector, and plasmids for in vitro transcription can be constructed. The constructed plasmid is transformed into a host bacterium for culture and amplification, and the plasmid is extracted. The extracted plasmid is digested into linear molecules using enzymes. Using the prepared linearized plasmid molecule as a template, mRNA is prepared using in vitro transcription. An in vitro transcription (IVT) system typically includes a transcription buffer, nucleotide triphosphates (NTPs), RNase inhibitors, and polymerases. NTPs can be selected from, but are not limited to, natural and non-natural (modified) NTPs. Polymerases can be selected from, but are not limited to, T7 RNA polymerase, T3 RNA polymerase, and mutant polymerases. Cap structure analogs can be added during in vitro transcription to directly obtain mRNA with a cap structure; alternatively, a capping enzyme and dimethyltransferase can be used to add a cap structure to the mRNA after in vitro transcription. The obtained mRNA can be purified using methods conventional in the art, such as chemical precipitation, magnetic beading, affinity chromatography, etc.

[0047] The S protein mutant of the first aspect of the present invention can be directly used as an antigen to prepare vaccines.

[0048] The mRNA described in the third aspect of this invention can be prepared together with lipid compounds into liposomes or lipid nanoparticles encapsulating the mRNA, and then prepared into a vaccine.

[0049] Therefore, a fifth aspect of the present invention is to provide a vaccine composition comprising the S protein mutant described in the first aspect of the present invention, or comprising the mRNA described in the third aspect of the present invention.

[0050] According to the present invention, in addition to containing an S protein mutant or mRNA, a lipid compound for forming liposomes or lipid nanoparticles, the vaccine or vaccine composition may also contain pharmaceutically acceptable excipients and / or immune adjuvants.

[0051] Lipid nanoparticles can be prepared using methods known in the art. For example, lipid molecules are dissolved in an organic solvent in a molar ratio to prepare a solution of mixed lipids. The solution of mixed lipids is used as the organic phase, and an aqueous solution of the delivered substance (e.g., nucleic acid) is used as the aqueous phase. The organic and aqueous phases are mixed to prepare lipid nanoparticles. Lipid nanoparticles can be prepared using methods including, but not limited to, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, nanoprecipitation, microfluidics, simple and complex coagulation, and other methods well known to those skilled in the art. The preparation method may further include the steps of separating and purifying the lipid nanoparticles. The preparation method may further include the step of lyophilizing the lipid nanoparticles.

[0052] According to the present invention, in the vaccine or vaccine composition, when lipid nanoparticles are used as carriers, mRNA is located in the lipid nanoparticles, and the lipid nanoparticles contain 30-60 mol% of ionizable / cationic lipid molecules, 5-30 mol% of neutral lipid molecules, 30-50 mol% of cholesterol lipid molecules, and 0.4-10 mol% of PEGylated lipid molecules, accounting for 30-60 mol% of their total lipid molecules; preferably, they contain 32-55 mol% of ionizable / cationic lipid molecules, 8-20 mol% of neutral lipid molecules, 35-50 mol% of cholesterol lipid molecules, and 0.5-5 mol% of PEGylated lipid molecules; more preferably, they contain 39-51 mol% of ionizable / cationic lipid molecules, 9-16 mol% of neutral lipid molecules, 37-49 mol% of cholesterol lipid molecules, and 1.3-2.7 mol% of PEGylated lipid molecules.

[0053] Ionizable / cationic lipid molecules can be selected from commercially available molecules such as DLin-MC3-DMA, DOTAP, and DOTMA. The ionizable lipid molecule represented by formula C is:

[0054] Formula C Each n3 is independent of each other and may be the same or different. Each n3 is selected from an integer from 1 to 8. Each m3 is independent of each other and may be the same or different. Each m3 is selected from an integer from 0 to 8. Preferably, each n3 is selected from an integer from 4 to 8, and each m3 is selected from an integer from 4 to 8. Preferably, each n3 is the same as each other, and each m3 is the same as each other.

[0055] In one embodiment of the present invention, n3 is preferably 6, m3 is preferably 4, and the molecular structure is as follows:

[0056]

[0057] Neutral lipid molecules can be selected from, for example, phosphatidylcholine compounds represented by formula E. E, Phosphatidylethanolamine compounds represented by formula F F, wherein Ra, Rb, Rc, and Rd are independently selected from straight-chain or branched C10-30 alkyl groups, straight-chain or branched C10-30 alkenyl groups, preferably CH3(CH2). 17 CH2-, CH3(CH2) 15 CH2-, CH3(CH2) 13 CH2-, CH3(CH2) 11 CH2-, CH3(CH2)9CH2-, CH3(CH2)7CH2-, CH3(CH2)7-CH=CH-(CH2)7-, CH3(CH2)4CH=CHCH2CH=CH(CH2)7-, CH3(CH2)7-CH=CH-(CH2)9-.

[0058] Cholesterol lipid molecules can be selected from cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate, for example.

[0059] PEGylated lipid molecules comprise a lipid moiety and a PEG-based polymer moiety, denoted as "lipid moiety-PEG-number-average molecular weight". The lipid moiety is a diacylglycerol or diacylglycerol amide, selected from dilauroylglycerol, dimyristoylglycerol, dipalmitoylglycerol, distearylglycerol, dilaurylglycerol amide, dimyristoylglycerol amide, dipalmitoylglycerol amide, distearylglycerol amide, 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine, and 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine. Amines; PEGs have a number average molecular weight of about 130 to about 50,000, for example about 150 to about 30,000, about 150 to about 20,000, about 150 to about 15,000, about 150 to about 10,000, about 150 to about 6,000, about 150 to about 5,000, about 150 to about 4,000, about 150 to about 3,000, about 300 to about 3,000, about 1,000 to about 3,000, about 1,500 to about 2,500, for example about 2,000.

[0060] In the vaccine composition, the mass ratio of total lipid molecules to mRNA is 5-20:1.

[0061] The application of the S protein mutant described in the first aspect of the present invention, or the mRNA described in the third aspect of the present invention, in the preparation of vaccines.

[0062] According to the present invention, the vaccine or vaccine composition may be used to prevent and / or treat 2019-nCoV infection or diseases or conditions related to 2019-nCoV infection, wherein the 2019-nCoV may be a wild-type strain or any mutant strain thereof. In one embodiment of the present invention, the 2019-nCoV is a B.1.617.2 mutant strain.

[0063] The diseases or conditions related to 2019-nCoV infection include, but are not limited to, pneumonia caused by 2019-nCoV infection, headache, nasal congestion, runny nose, cough and / or bronchitis caused by 2019-nCoV infection, disseminated intravascular coagulation caused by 2019-nCoV infection, and sepsis caused by 2019-nCoV infection.

[0064] The sixth aspect of the present invention is to provide the use of the DNA molecule described in the second aspect of the present invention in the preparation of S protein mutants, and the use of the nucleic acid molecule described in the fourth aspect of the present invention in the preparation of the mRNA described in the third aspect of the present invention.

[0065] Sequence list of this invention:

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073] The ionizable lipid compound of formula C in this invention can be synthesized using methods already known in the art, for example, by reacting an equivalent or more of an amine with an equivalent or more of an epoxy-terminated compound under suitable conditions. The synthesis of the ionizable lipid compound can be carried out with or without solvent, and the synthesis can be carried out at a higher temperature in the range of 25-100°C. The ionizable lipid compound can optionally be purified.

[0074] In some embodiments of the present invention, the ionizable lipid compounds of the present invention can be prepared using the following general preparation method.

[0075]

[0076] Step 1: Restore

[0077] In the presence of a reducing agent, the carboxyl group of compound A1 is reduced to a hydroxyl group to obtain compound A2. Examples of reducing agents include, but are not limited to, lithium aluminum hydride and diisobutylaluminum hydride. Examples of solvents used in the reaction include, but are not limited to, ethers (such as diethyl ether, tetrahydrofuran, and dioxane), halogenated hydrocarbons (such as chloroform, dichloromethane, and dichloroethane), hydrocarbons (such as n-pentane, n-hexane, benzene, and toluene), and mixtures of two or more of these solvents.

[0078] Step 2: Oxidation

[0079] In the presence of an oxidizing agent, the hydroxyl group of compound A2 is oxidized to an aldehyde group to obtain compound A3. Examples of oxidizing agents include, but are not limited to, 2-iodobenzoic acid (IBX), pyridine chlorochromate (PCC), pyridine dichlorochromate (PDC), Des Martin oxidant, manganese dioxide, etc. Examples of solvents used in the reaction include, but are not limited to, halogenated hydrocarbons (such as chloroform, dichloromethane, and dichloroethane), hydrocarbons (such as n-pentane, n-hexane, benzene, and toluene), nitriles (such as acetonitrile), and mixtures of two or more of these solvents.

[0080] Step 3: Halogenation-Reduction

[0081] First, under acidic conditions, the aldehyde α-hydrogen of compound A3 undergoes a halogenation reaction with a halogenating agent to obtain an α-haloaldehyde intermediate. Then, in the presence of a reducing agent, the aldehyde group of the α-haloaldehyde is reduced to a hydroxyl group to obtain compound A4. Examples of acidic conditions include, but are not limited to, DL-proline. Examples of halogenating agents include, but are not limited to, N-chlorosuccinimide (NCS) and N-bromosuccinimide (NBS). Examples of reducing agents include, but are not limited to, sodium borohydride, sodium cyanoborohydride, and sodium triacetoxyborohydride.

[0082] Step 4: Epoxidation

[0083] Compound A4 is subjected to an intramolecular nucleophilic substitution reaction in the presence of a base to obtain epoxide compound A5. Examples of bases include, but are not limited to, alkali metal hydroxides or hydrides, such as sodium hydroxide, potassium hydroxide, and sodium hydride. Examples of solvents used in the reaction include, but are not limited to, mixtures of dioxane and water.

[0084] Step 5: Ring-opening reaction

[0085] Compound A5 is reacted with an amine (e.g., N,N-di(2-aminoethyl)methylamine) via a ring-opening reaction to obtain the final compound. Examples of solvents used in the reaction include, but are not limited to, ethanol, methanol, isopropanol, tetrahydrofuran, chloroform, hexane, toluene, and diethyl ether.

[0086] The raw material A1 used in the preparation method can be commercially available or synthesized using conventional methods.

[0087] Terminology Explanation:

[0088] In this application, the terms novel coronavirus, 2019-nCoV, and SARS-CoV-2 have the same meaning.

[0089] In this specification and claims, conventional single-letter or three-letter codes for amino acid residues are used. Unless otherwise stated, amino acid sequences are written from left to right with the amino-to-carboxyl orientation.

[0090] For ease of reference, the S protein mutants of this invention are described using the following naming rules: original amino acid: position: substituted amino acid. According to these rules, for example, asparagine at position 30 being replaced by alanine is represented as: Asn30Ala or N30A; the absence of asparagine at the same position is represented as: Asn30* or N30*; the insertion of another amino acid residue, such as lysine, is represented as: Asn30AsnLys or N30NK; the absence of a continuous sequence of amino acid residues, such as the absence of amino acid residues 242-244, is represented as (242-244)* or Δ(242-244) or 242_244del; if, compared to other S protein parents, the S protein mutant contains a "deletion" and an insertion at that position, it is represented as: *36Asp or *36D, indicating a deletion at position 36 and the insertion of asparagine. When one or more optional amino acid residues can be inserted at a given position, it is represented as: N30A,E, or N30A or N30E. Furthermore, when this document identifies a suitable position for modification without suggesting any specific modification, it should be understood that any amino acid residue can substitute for the amino acid residue at that position. Therefore, for example, when modification of asparagine at position 30 is mentioned but not specified, it should be understood that the asparagine can be omitted or substituted with any other amino acid, i.e., any one of R, D, A, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V. In addition, "N30X" refers to any of the following substitutions: N30R, N30D, N30C, N30Q, N30E, N30G, N30H, N30I, N30L, N30K, N30M, N30F, N30P, N30S, N30T, N30W, N30Y, or N30V; or abbreviated as: N30R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.

[0091] Domain: As used herein, when referring to a polypeptide, the term “domain” refers to a motif of a polypeptide that has one or more identifiable structural or functional features or properties (e.g., binding capacity, serving as a site for protein-protein interactions).

[0092] The terms "protein mutant" or "peptide mutant" refer to molecules whose amino acid sequence differs from the native or reference sequence. Compared to the native or reference sequence, amino acid sequence mutants may have substitutions, deletions, and / or insertions at certain positions within the amino acid sequence. Typically, mutants will have at least approximately 50% identity with the native or reference sequence, at least approximately 60% identity, at least approximately 70% identity, at least approximately 80% identity, at least approximately 90% identity, at least approximately 95% identity, or at least approximately 99% identity.

[0093] In this specification and claims, nucleotides are referred to by their generally accepted single-letter codes. Unless otherwise stated, nucleotide sequences are written from left to right in a 5' to 3' orientation. Nucleotides are represented herein by generally known single-letter symbols recommended by the IUPAC-IUB Committee on Biochemical Nomenclature. Thus, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil. Those skilled in the art will understand that the T base in the codons disclosed herein exists in DNA, while the T base is replaced by a U base in the corresponding RNA. For example, the codon-nucleotide sequences in DNA form disclosed herein, such as vectors or in vitro translation (IVT) templates, have their T bases transcribed as U bases in their corresponding transcribed mRNAs. In this respect, codon-optimized DNA sequences (containing T) and their corresponding mRNA sequences (containing U) are both considered codon-optimized nucleotide sequences of this disclosure. Those skilled in the art will also understand that equivalent codon maps can be produced by replacing one or more bases with non-natural bases.

[0094] The terms "nucleic acid sequence," "nucleotide sequence," or "polynucleotide sequence" are used interchangeably and refer to a continuous nucleic acid sequence. The sequence can be single-stranded or double-stranded DNA or RNA, such as mRNA.

[0095] "A nucleotide sequence encoding..." refers to a nucleic acid (e.g., mRNA or DNA molecule) coding sequence that encodes a polypeptide. The coding sequence may further include start and stop signals operatively linked to regulatory elements, including promoters and polyadenylation signals capable of directing expression in the cells of an individual or mammal administering the nucleic acid.

[0096] Homology: As used herein, the term "homology" refers to the overall correlation between polymer molecules, for example, between nucleic acid molecules (e.g., DNA and / or RNA molecules) and / or between polypeptide molecules. Generally, the term "homology" implies an evolutionary relationship between two molecules. Therefore, two homologous molecules will share a common evolutionary ancestor. In the context of this disclosure, the term homology includes both identity and similarity.

[0097] In some embodiments, polymer molecules are considered “homological” if at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the monomers in the molecule are identical (completely identical monomers) or similar (conservative substitutions). The term “homological” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

[0098] Identity: As used herein, the term “identity” refers to the overall monomer conservation between polymer molecules, such as between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. For example, the percentage identity of two polynucleotide sequences can be calculated by aligning them for optimal comparison purposes (e.g., vacancies can be introduced in one or both of the first and second nucleic acid sequences for optimal alignment, and dissimilar sequences can be omitted for comparison purposes). In some embodiments, the length of the sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. Nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percentage identity between two sequences is a function of the number of common positions shared by the sequences, taking into account the number of vacancies that need to be introduced to achieve optimal alignment of the two sequences and the length of each vacancy. Sequence comparison and determination of the percentage identity between two sequences can be accomplished using mathematical algorithms. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent.

[0099] Suitable software programs are available from various sources and can be used for the alignment of both protein and nucleotide sequences. For example, a suitable program for determining percentage sequence identity is Bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Other suitable programs include Needle, Stretcher, Water, or Matcher, part of the bioinformatics EMBOSS program suite, and also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa. Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (ClustalW, ClustalX, or Clustal Omega), MUSCLE, etc.

[0100] The terms "coding region" and "coding area" refer to open reading frames (ORFs) in polynucleotides that, when expressed, produce polypeptides or proteins.

[0101] "Operationally linked" refers to a functional link between two or more molecules, constructs, transcripts, entities, parts, etc.

[0102] Expression: As used herein, “expression” of a nucleic acid sequence means one or more of the following events: (1) the generation of an mRNA template from a DNA sequence (e.g., by transcription); (2) the processing of the mRNA transcript (e.g., by splicing, editing, 5' cap formation and / or 3' end processing); (3) the translation of mRNA into a polypeptide or protein; and (4) post-translational modifications of the polypeptide or protein.

[0103] 5' Cap Structure: A 5' cap is typically a modified nucleotide (especially a guanine nucleotide) added to the 5' end of an mRNA molecule, but atypical cap analogs are also included. A 5' cap can be added using a 5'-5'-triphosphate bond (also known as m7GpppN). Other examples of 5' cap structures include glycerol groups, reverse deoxygenated debase residues (partially), 4',5'-methylene nucleotides, 1-(β-D-erythrofuranosyl) nucleotides, 4'-thionucleotides, carbocyclic nucleotides, 1,5-dehydrated hexitol nucleotides, L-nucleotides, α-nucleotides, modified base nucleotides, threopentafuranosyl nucleotides, acyclic 3',4'-closed nucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentayl nucleotides, 3'-3'-reverse nucleotide moiety, 3'-3'-reverse debase moiety, 3'-2'-reverse nucleotide moiety, 3'-2'-reverse debase moiety, 1,4-butanediol phosphate, 3'-aminophosphate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3'-thiophosphate, dithiophosphate, or bridged or non-bridged methylphosphonate moiety. These modified 5' cap structures can be used in the context of this invention to modify the mRNA sequence of this invention.

[0104] Cap analogs: Cap analogs are non-polymerizable dinucleotides that have a cap function because they facilitate translation or localization and / or prevent the degradation of RNA molecules when incorporated at the 5' end. Non-polymerizable means that cap analogs will only be incorporated at the 5' end because they do not have a 5' triphosphate and therefore cannot be extended in the 3' direction by template-dependent RNA polymerase. Cap analogs include, but are not limited to, chemical structures selected from the group consisting of: m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analogs (e.g., m2,7GpppG), trimethylated cap analogs (e.g., m2,2,7GpppG), dimethylated symmetrical cap analogs (e.g., m7Gpppm7G) or anti-reverse cap analogs (e.g., ARCA; m7,2'OmeGpppG, m7,2'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG and their tetraphosphate derivatives) (Stepinski et al., 2001. RNA 7(10):1486-95).

[0105] Naturally occurring or artificially synthesized nucleotide analogs, such as those selected from pseudouridine, 2-thiouridine, 5-methyluridine, 5-methylcytidine, N6-methyladenosine, N1-methylpseudouridine, 5-ethynyluridine, pseudo-UTP, N1-methyl-pseudo-UTP, 5-ethynyl-UTP, and 5-methyl-CTP.

[0106] "Pharmaceutically acceptable excipients" means any component other than the S protein mutant or mRNA described herein, and which has substantially non-toxic and non-inflammatory properties in patients, including but not limited to any and all solvents, dispersion media or other liquid carriers, dispersing or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, binders, lubricants, antioxidants, diluents, granulators and / or dispersants, antimicrobial or antifungal agents, molar osmolality regulators, pH regulators, colorants, sweeteners or flavorings, stabilizers, buffers, chelating agents, cryoprotectants and / or fillers, as appropriate for the desired specific dosage form. Various excipients used to formulate pharmaceutical compositions and techniques used to prepare compositions are known in the art. Exemplary antimicrobial or antifungal agents include, but are not limited to, benzalkonium chloride, benzyl chloride, methylparaben, ethylparaben, benzoic acid, hydroxybenzoic acid, potassium or sodium benzoate, potassium or sodium sorbate, sodium propionate, sorbic acid, etc., and combinations thereof. Exemplary preservatives include, but are not limited to, beta-carotene, citric acid, ascorbic acid, butylated hydroxyanisole, sodium lauryl sulfate (SLS), vitamin A, vitamin C, vitamin E, sodium lauryl ether sulfate (SLES), and combinations thereof. Exemplary buffers for controlling pH may include, but are not limited to, sodium phosphate, sodium succinate, histidine (or histidine-HCl), sodium malate, sodium citrate, sodium carbonate, and / or combinations thereof. Exemplary cryoprotectants include, but are not limited to, trehalose, lactose, glycerol, mannitol, sucrose, dextrose, and combinations thereof. Exemplary fillers may include, but are not limited to, mannitol, glycine, lactose, sucrose, trehalose, raffinose, and combinations thereof.

[0107] "And / or" will be considered as a specific disclosure of each of the two specified features or components having or not having the other. Therefore, the term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0108] "Comprising" and "including" have the same meaning and are intended to be open and allow, but do not require, the inclusion of additional elements or steps. When the terms "comprising" or "including" are used herein, the terms "consisting of" and / or "substantially consisting of" are also included and disclosed.

[0109] “Approximately”: The term “approximately” used in conjunction with numerical values ​​throughout the specification and claims indicates a range of accuracy familiar and acceptable to those skilled in the art. Typically, this range of accuracy is ±10%. Attached Figure Description

[0110] Figure 1 This diagram illustrates the primary structure of the 2019-nCoV S protein and its conformation before prefusion. Part A of the diagram shows the primary structure of the S protein: SS (signal sequence), NTD (N-terminal domain), RBD (receptor binding domain), S2' (S2' protease cleavage site), FP (fusion peptide), HR1 (heptad repeat 1), CH (central helix), CD (connector domain), HR2 (heptad repeat 2), TM (transmembrane domain), CT (cytoplasmic tail), and the arrows indicate protease cleavage sites. The S1 subunit precedes S1 / S2, and the S2 subunit follows. Part B of the diagram shows the side and top views of the S protein structure before prefusion.

[0111] Figure 2 : A statistical graph showing the intracellular protein expression levels of mRNA prepared from different in vitro transcription vectors using Firefly Luc as a reporter protein.

[0112] Figure 3 The integrity of B.1.617.2 mRNA was analyzed using an RNA 6000 nano chip on a 2100 bioanalyzer.

[0113] Figure 4 ELISA was used to detect the expression level of the S protein mutant in the supernatant after nucleic acid transfection of CHO-K1 cells.

[0114] Figure 5 : A three-dimensional structural model of the S protein mutant of this invention.

[0115] Figure 6 The expression level of S protein in the supernatant after cell transfection with the mRNA of the present invention was encapsulated by lipid nanoparticles prepared by II-37 (also known as C2).

[0116] Figure 7 BALB / c mouse immunization strategy diagram

[0117] Figure 7-1 Results of specific IgG binding antibody detection in BALB / c mice immunized with mRNA vaccine. BALB / c mice (n=4) were intramuscularly injected with different doses of vaccine or phosphate-buffered saline (PBS, control group, n=4) on days 0 and 21. Blood samples were collected on day 35, and the concentration of SARS-CoV-2 B.1.617.2 strain S protein-specific IgG binding antibody in the blood samples was determined by ELISA. Each dot represents a single animal, dots with the same value are covered, and the numbers shown in the figure are median values.

[0118] Figure 7-2 Results of ACE2 competitive inhibition assay after mRNA vaccine immunization in BALB / c mice. BALB / c mice (n=4) were intramuscularly injected with different doses of the vaccine or phosphate-buffered saline (PBS, control group, n=4) on days 0 and 21. Blood samples were collected on day 35, and the titer of neutralizing antibodies that competitively bind to the S protein of SARS-CoV-2 strain B.1.617.2 was measured. Results are expressed as inhibition rate (%). The numbers shown in the figure are median values, and 20% is the inhibition rate cut-off value.

[0119] Figure 7-3 Results of pseudovirus neutralizing antibody detection in BALB / c mice immunized with mRNA vaccine. BALB / c mice (n=4) were intramuscularly injected with different doses of the vaccine or phosphate-buffered saline (PBS, control group, n=4) on days 0 and 21. Blood samples were collected on day 35, and pVNT50 of the SARS-CoV-2 B.1.617.2 strain was determined using the reporter gene assay (Vazyme). The numbers shown in the figure are median values.

[0120] Figure 8 Schematic diagram of rhesus monkey immunization strategy

[0121] Figure 8-1 Results of specific IgG binding antibody detection in rhesus monkeys immunized with mRNA vaccine. Female and male rhesus monkeys (9–22 years old) were intramuscularly injected with 10 μg, 30 μg, or 100 μg of mRNA vaccine on days 0 and 28 (n=3), while the control group was injected with saline (n=2). Blood samples were collected on day 35, and the concentration of IgG binding antibodies specific to the S protein of SARS-CoV-2 strain B.1.617.2 was measured by ELISA. The figures shown in the figure are median values.

[0122] Figure 8-2Results of ACE2 competitive inhibition assay in rhesus monkeys after mRNA vaccine immunization. Female and male rhesus monkeys (9–22 years old) were intramuscularly injected with 10 μg, 30 μg, or 100 μg of mRNA vaccine on days 0 and 28 (n=3), while the control group was injected with saline (n=2). Blood samples were collected on day 35, and the titer of neutralizing antibodies that competitively bind to the S protein of strain B.1.617.2, which binds to ACE2, was measured. Results are expressed as inhibition rate (%). The numbers shown in the figure are median values, and 20% is the cut-off value.

[0123] Figure 8-3 Results of pseudovirus neutralizing antibody detection in rhesus monkeys after mRNA vaccine immunization. Female and male rhesus monkeys (9–22 years old) were intramuscularly injected with 10 μg, 30 μg, or 100 μg of mRNA vaccine on days 0 and 28 (n=3), while the control group was injected with saline (n=2). Blood samples were collected on day 35, and pVNT50 of SARS-CoV-2 strain B.1.617.2 was determined using the reporter gene assay (Vazyme). The figures shown in the figure are median values.

[0124] Figure 9 Immunization strategy diagram for H11 K18-hACE2 transgenic mice

[0125] Figure 9-1 Results of specific IgG binding antibody detection in H11 K18-hACE2 transgenic mice after immunization with mRNA vaccine. Mice (n=10) were intramuscularly injected with different doses of mRNA vaccine or saline on days 0 and 25 (control group, n=10); the challenge control group (n=8) did not receive injection. Blood samples were collected on day 32, and the concentration of IgG binding antibody specific to the S protein of SARS-CoV-2 B.1.617.2 strain in the blood samples was determined by ELISA. Each point represents a single animal, and points with the same value were covered. The numbers shown in the figure are medians. P-values ​​were analyzed by one-way ANOVA (ns, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0126] Figure 9-2 Results of neutralizing antibody titers in H11 K18-hACE2 transgenic mice immunized with mRNA vaccine. Blood samples were collected on day 32, and the titers of neutralizing antibodies that competitively bind to the S protein of strain B.1.617.2 against ACE2 were measured. Results are expressed as inhibition rates. The numbers shown in the figure are median values, and 20% is the inhibition rate cut-off value.

[0127] Figure 10Statistical chart of viral load in various tissues during challenge experiment in H11 K18-hACE2 transgenic mice after immunization with mRNA vaccine. Detailed Implementation

[0128] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0129] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. The experimental methods described are conventional molecular biology methods in the art and can be performed with reference to molecular biology lab manuals or kit product instructions.

[0130] Example 1: Efficiency Comparison Experiment of the IVT Vector of the Present Invention

[0131] In this embodiment, Firefly Luc was used as a reporter protein. Different IVT vectors were constructed to synthesize mRNA capable of translating Firefly Luc in vitro, and the translation efficiency of synthesized mRNAs with different sequence characteristics was compared.

[0132] Using conventional plasmid vector construction techniques in the field, the coding sequence of Firefly Luc was cloned into the multiple cloning site of the corresponding vectors, resulting in vectors numbered IVT1, IVT2, IVT3, and IVT4, respectively. Then, the corresponding Firefly Luc mRNA samples were prepared by in vitro transcription using the T7 in vitro transcription kit (cat#AM1344, purchased from Thermo Fisher Scientific) based on the aforementioned vectors.

[0133] Vectors IVT1 through IVT4 were all modified from the commercial vector psp73. The following sequences were inserted at the XhoI / NdeI restriction site of vector psp73. IVT1 did not include a UTR sequence, and the polyA tail was 64 alphas long. IVT2 used the 5' UTR shown in SEQ ID NO:6 and the 3' UTR sequence of GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC (the 3' UTR sequence of β-globin), with a polyA length of 120 alphas. IVT3 used the 5' UTR shown in SEQ ID NO:6 and the 3' UTR sequence shown in SEQ ID NO:7, with a polyA length of 120 alphas. IVT4 used the 5' UTR shown in SEQ ID NO:6 and two tandem repeats of SEQ ID NO:6. The 3'UTR sequence shown in NO:7 has a polyA length of 120 A's. Multiple cloning sites containing the common restriction enzyme sites HindIII and EcoRI were inserted between the 5'UTR and 3'UTR sequences. The coding sequence of Firefly Luc was then cloned into the HindIII and EcoRI multiple cloning sites. All vectors were constructed by GenScript using gene synthesis methods.

[0134] Each Firefly Luc mRNA sample was transfected into CHO cells using Lipofectamine 2000 (cat #11668030, purchased from Thermo Fisher Scientific) as the transfection reagent, and then transfected using Dual-Lumi. TM A dual-luciferase reporter gene assay kit (at#RG088S, purchased from Shanghai Beyotime Biotechnology Co., Ltd.) was used to detect luciferase. Firefly Luc DNA was transformed into the psicheck2 plasmid as a positive control (psicheck2 plasmid, cat#60908-6151, purchased from Beijing Tianenze Gene Technology Co., Ltd.). The specific steps were as follows: On day one, CHO cells were seeded into 96-well plates at 1.5 × 10⁶ cells per well. 4Cells were cultured overnight in F12K + 10% FBS. On the second day, before transfection, the medium was changed to serum-free F12K medium, and mRNA or DNA was transfected into CHO cells using Lipofectamine 2000. The amount of nucleic acid used per well was 100 ng, the amount of liposomes used was 0.3 μl, and the total volume of each well was 100 μl. The cells were cultured overnight. On the third day, the serum-free medium was changed to complete medium (F12K + 10% FBS), and the cells were cultured for another 24 hours. On the fourth day (48 hours after transfection), the Firefly Luc fluorescence value was detected.

[0135] The results are as follows Figure 2 As shown in the figure. "DNA" is the positive control (psicheck2 plasmid carrying the Firefly Luc gene), "IVT1-Luc", "IVT2-Luc", "IVT3-Luc", and "IVT4-Luc" represent the corresponding Firefly Luc mRNAs transcribed in vitro from vectors of IVT1, IVT2, IVT3, and IVT4, respectively, and "Nagative control" is the negative control. Figure 2 It is evident that, under the same mRNA transfection level, the protein expression level of IVT4-Luc is much higher than that of the other three mRNAs, by 2-3 times, indicating that IVT4-Luc has good stability and high translation efficiency.

[0136] Example 2 B.1.617.2 Preparation and Translation of mRNA

[0137] 1. A nucleic acid sequence encoding the mRNA shown in SEQ ID No. 8 is artificially synthesized and cloned after the T7 promoter of the pUC57-kana vector, which has been previously modified to contain sequences encoding SEQ ID NO: 6, the Kozak sequence, two consecutive SEQ ID NO: 7 sequences, and a polyA tail. The nucleic acid sequence encoding the mRNA shown in SEQ ID NO: 8 is cloned at the multiple cloning site between the Kozak sequence and the two consecutive SEQ ID NO: 7 sequences to construct a plasmid for in vitro transcription.

[0138] 2. Transform the constructed plasmid into E. coli Dh5a, culture and amplify, and extract the plasmid.

[0139] 3. The extracted plasmid was digested into linear molecules using the restriction endonuclease SpeI, which is located immediately after the polyA tail.

[0140] 4. Using the prepared linearized plasmid molecule as a template, mRNA was prepared using in vitro transcription (Thermo's In Vitro Transcription Kit A45975). The sequence of the mRNA is shown in SEQ ID NO:9, and will be referred to as B.1.617.2 mRNA below. The S protein mutant obtained after translation from this mRNA is the present invention, whose amino acid sequence from the N-terminus to the C-terminus is a direct link between the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:3. After in vitro transcription, a CAP1 cap structure was added to the mRNA using a capping enzyme and dimethyltransferase.

[0141] 5. Purification of mRNA: The obtained mRNA stock solution was purified using affinity chromatography.

[0142] 6. mRNA quality control: The prepared mRNA was analyzed for integrity using an RNA 6000 nanochip on a 2100 bioanalyzer. The results are as follows: Figure 3 As shown, the transcribed mRNA band was single and showed no obvious degradation.

[0143] In addition, the Spike fragment was excised from the commercially available plasmid pCMV3-spike using the restriction endonucleases HindIII and EcoRI, and inserted between the HindIII and EcoRI sites of the IVT1 vector in Example 1 to obtain the IVT1-spike plasmid. This plasmid was then point-mutated to obtain the IVT1-spike-D614G plasmid. Using this plasmid as a template, spike-D614G mRNA was transcribed in vitro to express the full-length S protein containing the D614G mutation.

[0144] 7.B.1.617.2 Detection of mRNA expression at the cellular level: Using the CHO-K1 cell line as the expression system, mRNA was transfected using Lipofectamine Messenger MAX Reagent (Invitrogen, Cat#1168-027). After culturing for 48 h, the cell culture supernatant was collected, and the expression level of S protein was detected using an enzyme-linked immunosorbent assay (ELISA) kit to assess whether the mRNA could be translated into protein. Results are as follows: Figure 4 As shown. Figure 4In this text, "spike DNA" refers to the commercially available plasmid pCMV3-spike (purchased from Sinocare), which expresses the full-length wild-type S protein; "spike-D614G mRNA" refers to the aforementioned mRNA expressing the full-length S protein containing the D614G mutation; and "spike B.1.617.2 mRNA" refers to the aforementioned B.1.617.2 mRNA, which expresses the S protein mutant described in this invention. The results indicate that the mRNA of this invention can highly express the S protein mutant in cells.

[0145] After purification, the obtained S protein mutant was structurally analyzed using cryo-electron microscopy. The 3D structure of the S protein is shown below. Figure 5 As shown, the S protein mutant exhibits a stable prefusion spike structure. The B.1.617.2 mutant strain differs from the wild-type strain in nine mutation sites, two of which are in the RBD region. Previously reported wild-type prefusion S protein RBD regions primarily exhibit a structure of one open and two closed segments. The S protein mutant of this invention exhibits a flexible structure primarily of two open and one closed segments. This structural difference forms the basis for enhanced viral binding to the ACE2 receptor and increased infectivity. Furthermore, this structural difference leads to significant differences in the immunogenic epitopes of the S protein, resulting in significant differences in antibodies, especially neutralizing antibodies, induced by different structures.

[0146] Example 3: Construction of LNP-loaded mRNA

[0147] Nanoparticles encapsulating mRNA were prepared using II-37 (also known as C2) as an ionizable lipid. Compounds II-37, DSPC, CHOL, and DMG-PEG2000 were accurately weighed and dissolved thoroughly in anhydrous ethanol in a suitable container. The specific molar ratio was: II-37:DSPC:CHOL:DMG-PEG2000 = 45:15:38.5:1.5. The lipid solutions were mixed uniformly in the specified ratio to form the organic phase. The B.1.617.2 mRNA from Example 2 was prepared as an aqueous solution (using pure water as the solvent) with a pH of 4.

[0148] An organic phase and an aqueous phase were mixed in a 3:1 volume ratio to prepare a lipid nanoparticle suspension on a microfluidic platform (e.g., PNI Ignite). The resulting lipid nanoparticle suspension was then purified and concentrated by centrifugation in a 100 kDa ultrafiltration centrifuge tube, and the concentrated liquid was dispensed.

[0149] The prepared lipid nanoparticles were measured for particle size, PDI, and potential using a laser nanoparticle size analyzer, and the encapsulation efficiency (EE%) was measured using a UV spectrophotometer combined with a RiboGreen RNA kit. Some samples were transfected into A549 cells according to the method in Example 2, and the cell transfection rate was detected by ELISA.

[0150] The physicochemical quality control data of the prepared lipid nanoparticles are shown in the table below:

[0151] mRNA-LNP 147.8±20.6 0.0651 34.28 100％

[0152] As a control, liposomes were also prepared by encapsulating B.1.617.2 mRNA from Example 2 with LipofectamineMax, and both liposomes and the above-mentioned lipid nanoparticles were transfected into A549 cells. The negative control was lipid nanoparticles prepared from II-37 without mRNA. The results are as follows: Figure 6 As shown, after the lipid nanoparticles carrying mRNA were transfected into cells, the protein expression level in the cells was much higher than that of the control reagent LipofectamineMax, indicating that the cell transfection efficiency of the prepared lipid nanoparticles was very high.

[0153] Example 4: Determination of the immunogenicity of the S protein mutant

[0154] The mRNA vaccine used was the same as the LNP lipid nanoparticles loaded with mRNA prepared in Example 3, with a molar ratio of lipid component II-37:DSPC:CHOL:DMG-PEG2000 of 45:15:38.5:1.5. Experimental methods:

[0155] ELISA method for detecting specific IgG binding antibodies.

[0156] The content of 2019-nCoV-specific IgG antibodies in the plasma of immunized animals was detected by indirect ELISA. 0.05 μg of the Spike antigen protein of the 2019-nCoV B.1.617.2 mutant strain was coated onto an ELISA plate (Thermo, Catalog Number #442404) and incubated overnight at 2-8°C. The plate was blocked with 3% BSA (SIGMA, Catalog Number #A7030) for 1 h at room temperature, then incubated with diluted mouse plasma (1:50) and monkey plasma (1:500) for 2 h, followed by washing five times with PBST. Then, HRP-conjugated goat anti-mouse / monkey secondary antibody was added and incubated at room temperature for 30-45 min, followed by washing five times with PBST. The antibody was developed using TMB (Thermofisher, Catalog Number #34029) and incubated at room temperature for 7 min. The reaction was terminated by adding stop solution (Solarbio, Catalog Number #C1058), and the absorbance was measured at 450 nm to determine the antibody content. A standard curve was fitted using a polynomial method with positive antibodies (mouse group: Yiqiao Shenzhou Cat#40591-MM43; rhesus monkey group: ACRO Cat#SPD-M201) to determine the total antibody amount.

[0157] The ACE2 binding inhibition assay is used to detect the competitive binding of neutralizing antibodies to antigen proteins in samples using an ELISA method.

[0158] Using the ELISA Anti-SARS-CoV-2 Neutralizing Antibody Titer Serologic Assay Kit (ACRO, Catalog Number #RAS-N031 / RAS-N040 / RAS-N056), mouse / monkey plasma was diluted and added to microplates pre-coated with Human ACE2 Protein. The neutralizing antibody against the 2019-nCoV B.1.617.2 mutant strain in the plasma (Spike RBD) specifically competed with the pre-coated Human ACE2 Protein on the microplate for HRP-SARS-CoV-2 Spike. After incubation at 37°C for 1 h, the plate was incubated with substrate at 37°C for 20 min for color development, followed by termination with stop solution. The absorbance of the samples was measured at 450 nm / 630 nm using a BioTek ELISA reader (SLXFATS). The OD450 nm reading was subtracted from the OD630 nm reading to reduce background interference. Inhibition rate calculation method: OD450nm inhibition rate = (1 - sample OD450 nm / Negative Control OD450 nm) × 100%.

[0159] Pseudovirus neutralization assay (reporter gene method)

[0160] Neutralizing antibodies can block the binding of the S protein and ACE2 on the surface of SARS-CoV-2 pseudovirus, thereby preventing the pseudovirus from infecting host cells. The degree of viral blockage can be inferred by detecting the expression level of the reporter gene luciferase. Plasma / serum samples were collected from mice / monkeys at different time points before and after vaccine injection. All samples were heat-inactivated by incubating in a 56°C water bath for 30 min before use. The samples were diluted 20-fold with serum-free DMEM (Gibco Catalog Number #C11995500CP) medium and filtered through a 0.22 μm filter membrane for sterilization. Then, three-fold serial dilutions were prepared in DMEM medium containing 10% FBS (Gibco Catalog Number #10099-141C), resulting in six gradients. The SARS-CoV-2-Fluc pseudovirus (Vazyme) was thawed in advance by transferring it from -80°C to 4°C or on ice. Before use, the virus was diluted to 1-2 × 10⁻⁶ with DMEM medium containing 10% FBS serum. 4 TCID50 / ml. Mix the virus suspension with an equal volume of plasma in a 96-well plate and incubate at 37°C for 1 h. Add 50 μL of the solution at a density of 2 × 10⁻⁶ ml to each well. 4 After culturing 293 cells overexpressing ACE2 in cells / wells for 48 h, 100 μL of culture medium was aspirated from the 96-well plate, and 100 μL of Bio-Lite reporter gene (Vazyme, Catalog Number #DD1201) assay reagent was added after equilibration at room temperature. The plate was shaken for 2 min, incubated at room temperature for 5 min, and then the chemiluminescence value (RLU) was detected using a multi-functional microplate reader (TECAN, Spark).

[0161] The inventors’ previous research has confirmed that both the ACE2 competitive inhibition method and the pseudovirus neutralization method can effectively characterize the degree of neutralization of live virus in rhesus monkey experiments, and have important reference value for judging the immunogenicity of vaccines.

[0162] The BALB / c mice used in this experiment were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Animal Production License: SCXK(Jing)2021-0006), 6-8 weeks old, and female BALB / c mice (SPF level) were used for the experiment. The H11-K18-hACE2 transgenic mice were purchased from Jiangsu Jicui Yakang Biotech Co., Ltd. (Production Animal License: SCXK(Su)2018-0008), 6 weeks old, SPG level; this ACE2 humanized mouse model was developed by humanizing ACE2 on the C57BL / 6JGpt background mouse, and the human cytokeratin 18 (Cytokeratin 18, K18) promoter was used to drive the overexpression of hACE2 at the safe island H11 locus, which was used to simulate the phenotype of severe COVID-19 in humans. The rhesus monkeys were 9-22 years old, both male and female, healthy. During the environmental adaptation and quarantine period, their appearance, mental state, posture, respiration, fecal and urinary conditions, and food and water intake were examined and found to be normal, meeting the experimental requirements.

[0163] 1. Immunogenicity test of mRNA vaccine in BALB / c mice

[0164] The immunization strategy for BALB / c mice is shown in Figure 7 , with a 21-day interval between the two immunizations. Blood was collected routinely for antibody detection.

[0165] Six dose groups were set up in the experiment, and a PBS control group was set up separately. The exploration range of the six dose groups increased sequentially by 4 times from the lowest dose of 0.02 μg, namely 0.02, 0.08, 0.3, 1.25, 5 to the highest dose group of 20 μg.

[0166] Results:

[0167] The detection of specific IgG binding antibodies is as shown in Figure 7-1 . Compared with the PBS control group, all dose groups could significantly induce the production of specific IgG antibodies against the S protein of the B.1.617.2 strain. The median antibody concentration in the 20 μg highest dose group was 14802 ng / mL, and the median antibody concentrations in the 0.02 - 5 μg groups were 165, 1355, 4015, 1809, and 7234 ng / mL respectively, showing a dose-effect relationship.

[0168] The inhibition results of ACE2 competing with the S protein of the B.1.617.2 strain are as shown in Figure 7-2 , expressed as the inhibition rate (%). The results showed that from the 0.08 μg group, 0.3 μg group, 1.25 μg group, 5 μg group to the 20 μg group, the median inhibition rates were 58%, 80%, 79%, 90% and 91% respectively. Among them, the inhibition rate in the 20 μg high dose group was above 91%.

[0169] The results of pseudovirus neutralizing antibody levels are as follows: Figure 7-3 As shown in the figure, starting from the 0.08 μg group, all groups were able to induce a relatively high level of neutralizing antibodies.

[0170] 2. Preliminary immunogenicity test of mRNA vaccine in rhesus monkeys

[0171] See rhesus monkey immunization strategy. Figure 8 Two immunizations were administered 28 days apart, with routine blood sampling for antibody testing.

[0172] The experiment included three dosage groups: 10, 30, and 100 μg, representing low, medium, and high doses, respectively. A separate control group was also established using physiological saline.

[0173] Specific IgG binding antibody detection, such as Figure 8-1 As can be seen, compared with the saline control group, all dosage groups could induce the production of S protein-specific IgG antibodies against strain B.1.617.2. The median antibody concentrations in the high, medium, and low dosage groups were 141,553, 63,249, and 82,458 ng / mL, respectively.

[0174] The results of ACE2 competitive binding to the S protein of strain B.1.617.2 are as follows: Figure 8-2 The inhibition rate was expressed as a percentage. The results showed that the median inhibition rates of ACE2 competitive binding in rhesus monkeys were 89%, 88%, and 98% in the 10 μg, 30 μg, and 100 μg dose groups, respectively.

[0175] The results of pseudovirus neutralizing antibody levels are as follows: Figure 8-3 The GMT values ​​for the high, medium, and low dose groups were 3000, 392, and 434, respectively.

[0176] Antibody levels in rhesus monkeys showed a dose-dependent effect, meaning that increasing the vaccine dose induced dose-dependent humoral and cellular immune responses.

[0177] Mice can produce high levels of neutralizing antibodies as low as 0.08 μg, while rhesus monkeys produce highly efficient antibodies at a minimum of 10 μg.

[0178] 3. Efficacy and challenge tests of mRNA vaccine in H11 K18-hACE2 transgenic mice.

[0179] The immunization strategy for H11 K18-hACE2 transgenic mice is as follows: Figure 9 The two immunizations were administered 25 days apart. Blood was routinely drawn for antibody testing, and the patients were transferred to a P3 laboratory for challenge experiments 14 days after the second immunization.

[0180] The experiment included three dosage groups, along with a separate saline control group and a blank mouse control group. The 0.8 μg, 4 μg, and 20 μg groups were designated as low, medium, and high dose groups, respectively. The challenge control group received no injection.

[0181] Specific IgG binding antibody detection by Figure 9-1 As can be seen, compared with the saline control group, all dosage groups significantly induced the production of S protein-specific IgG antibodies against strain B.1.617.2. The median antibody concentration in the highest dose group (20 μg) was 2621 ng / mL, while the median antibody concentrations in the 4 μg and 0.8 μg groups were 1121 and 155 ng / mL, respectively. There was no statistically significant difference between these groups and the 20 μg group, indicating a dose-response relationship.

[0182] The results of ACE2 competitive binding to the S protein of strain B.1.617.2 are as follows: Figure 9-2 The inhibition rate was used as the metric. The results showed that different dose groups exhibited different inhibitory effects due to individual differences.

[0183] B.1.617.2 Virus challenge test: Mice were challenged by intranasal administration of virus suspension at a volume of 20 μl / mouse and a challenge dose of 1000 TCID. 50 The observation period after challenge was 5 days. On day 3 (3 dpi) and day 5 (5 dpi) after challenge, 4 animals were euthanized per instance in the blank control group, and 5 animals were euthanized per instance in each challenge group. Lung, brain, intestinal tissue, heart, liver, kidney and spleen were collected, and the viral load in each tissue was detected by qPCR. The above tissues were also collected for pathological HE analysis.

[0184] Compared with the challenge control group 2, the viral load in the lung tissue of each immunization group (low-dose group #3, medium-dose group #4, and high-dose group #5) decreased significantly on both days 3 and 5 post-infection, with a decrease exceeding 2 Log10 values. Similarly, the viral load in the lung tissue of the low-dose group (#3) decreased by more than 2 Log10 values ​​on day 5 post-infection. The viral load in the brain tissue of each immunization group (#3, #4, and #5) decreased significantly on both days 3 and 5 post-infection, with a decrease exceeding 2 Log10 values. Compared with the challenge control group 2, the viral load in the heart, liver, spleen, kidney, and intestinal tissues of each immunization group (#3, #4, and #5) showed varying degrees of decrease at both time points, with some tissues showing a decrease exceeding 2 Log10 values ​​at some time points. Figure 10 ).

[0185] Histopathological (HE) changes: In the challenge control group, all 10 mice showed moderate to severe lung lesions, with widened and congested pulmonary septa and inflammatory cell infiltration. The high, medium, and low dose immune groups showed mild to moderate lesions, with no severe lesions, and the incidence of moderate lesions was lower than in the challenge control group. Comparison of the severity of heart and spleen lesions revealed varying degrees of reduction in the high, medium, and low dose immune groups compared to the challenge control group. The lesions in other organs (liver, intestines, and brain) were relatively mild.

[0186] In summary, in the SARS-CoV-2 B.1.617.2 strain-infected H11 K18-hACE2 transgenic mouse model, the weight changes in the challenge control group were consistent with clinical manifestations. At 5 days post-infection (dpi), the weight changes in each immunization group (3#, 4#, 5#) showed varying degrees of improvement, with the high-dose group (5#) showing weight gain. Viral load in the lungs and brain tissue decreased significantly. At 3 and 5 days post-infection, the viral load in the lungs of the high-dose group (5#) decreased by more than 2 Log10 values. At 5 days post-infection, the viral load in the lungs of the low-dose group (3#) showed a significant difference, decreasing by more than 2 Log10 values. The viral load in the brain tissue of each immunization group also showed significant differences, decreasing by more than 2 Log10 values. Viral load in other tissues decreased to varying degrees. Pathological changes in lung tissue were improved to varying degrees in each immunization group (3#, 4#, 5#) compared to the control group. Therefore, the mRNA vaccine against SARS-CoV-2 B.1.617.2 strain has a protective effect against SARS-CoV-2 B.1.617.2 strain infection in H11-K18-hACE2 transgenic mice.

[0187] Example 5: Synthesis of ionizable lipid compound II-37 of formula C

[0188]

[0189] Synthesis of linolenic acid (a2): LiAlH4 (7.20 g) and linolenic acid (50 g, a1) were added to 950 mL of tetrahydrofuran at 0 °C. The mixture was then stirred at 25 °C for 2 h. Thin-layer chromatography (TLC) showed that after the reaction was complete, water (7.2 mL), NaOH aqueous solution (7.2 mL, 15% by mass), and water (21.6 mL) were added sequentially to quench the reaction mixture. A suitable amount of Na2SO4 was added and the mixture was stirred for 15 minutes. The mixture was then filtered through a Buchner funnel, and the filter cake was washed with ethyl acetate. The filtrate was collected and concentrated by evaporation to obtain 47.4 g of the target product, linolenic acid (a2).

[0190] 1H NMR (400MHz, CDCl3): δ5.27-5.44(m,4H),3.63(t,J=6.63Hz,2H),2.77(t,J=6.44H z,2H),1.97-2.12(m,4H),1.57-1.63(m,1H),1.20-1.46(m,18H),0.83-0.95(m,3H)

[0191] Synthesis of (9Z,12Z)-octadec-9,12-dienal (a3): Linoleic acid (25.0 g, a2) and 2-iodobenzoic acid (39.4 g) were added to 170 mL of acetonitrile at room temperature, and the mixture was stirred at 85 °C for 4 h. The reaction solution was filtered through a Buchner funnel and the filter cake was washed with dichloromethane. The filtrate was collected and concentrated by evaporation to give 24.0 g of the target product (9Z,12Z)-octadec-9,12-dienal (a3).

[0192] 1 H NMR (400MHz, CDCl3): δ9.76(t,J=1.76Hz,1H),5.25-5.43(m,4H),2.76(t,J=6.17Hz,2H),2.41(td,J=7 .33,1.87Hz,2H),2.04(q,J=6.84Hz,4H),1.56-1.68(m,2H),1.22-1.36(m,14H),0.88(t,J=6.73Hz,3H)

[0193] Synthesis of (9Z,12Z)-2-chloro-octadec-9,12-dien-1-ol (a4): At 0 °C, (9Z,12Z)-octadec-9,12-dienal (43.0 g, a3), DL-proline (5.62 g), and N-chlorosuccinimide were added to 246 mL of acetonitrile, and the mixture was stirred at 0 °C for 2 h. After the reaction was complete, the reaction mixture was diluted with anhydrous ethanol (246 mL), and sodium borohydride (8.8 g) was added. The mixture was then stirred at 0 °C for 4 h. The reaction mixture was quenched with water (120 mL) and extracted with methyl tert-butyl ether. The combined organic phases were washed with saturated brine, dried over sodium sulfate, filtered, and concentrated by evaporation to obtain the target product (9Z,12Z)-2-chloro-octadec-9,12-dien-1-ol (a4, 46 g), which was used directly in the next step.

[0194] 1H NMR (400MHz, CDCl3): δ5.25-5.51(m,4H),3.97-4.07(m,1H),3.79(dd,J=12.01,3.63Hz,1H),3.59- 3.70(m,1H),2.67-2.90(m,2H),1.96-2.15(m,5H),1.64-1.82(m,1H),1.20-1.49(m,15H),0.89(br t,J=6.75Hz,3H)

[0195] Synthesis of 2-[(7Z,10Z)-hexadecane-7,10-diene]ethylene oxide (a5): At room temperature, (9Z,12Z)-2-chloro-octadecane-9,12-dien-1-ol (45 g, a4) and an aqueous solution of sodium hydroxide (120 g of sodium hydroxide dissolved in 585 mL of water) were added to 450 mL of 1,4-dioxane. After the addition was complete, the mixture was stirred at 35 °C for 2 h. After the reaction was completed by TLC, the reaction solution was separated by a separatory funnel, washed with saturated brine, dried with sodium sulfate, filtered, and concentrated by evaporation. The residue was then purified by rapid column chromatography with petroleum ether / ethyl acetate to obtain 29.11 g of the target product 2-[(7Z,10Z)-hexadecane-7,10-diene]ethylene oxide (a5).

[0196] 1 H NMR (400MHz, CDCl3): δ5.27-5.46(m,4H),2.87-2.98(m,1H),2.70-2.85(m,3H),2.4 6(dd,J=5.00,2.75Hz,1H),1.94-2.21(m,4H),1.24-1.58(m,17H),0.78-1.00(m,3H)

[0197] Synthesis of II-37: At room temperature, 2-[(7Z,10Z)-hexadecane-7,10-diene]ethylene oxide (5 g) and N,N-bis(2-aminoethyl)methylamine (739 mg) were added to 10 mL of ethanol, and the mixture was stirred at 90 °C for 36 h. The reaction solution was evaporated and concentrated, and the residue was purified by rapid column chromatography with dichloromethane / methanol elution to obtain crude product II-37 (4 g). The target product was further purified by rapid column chromatography with dichloromethane / methanol to obtain II-37 (2.2 g).

[0198] 1H NMR (400MHz, CDCl3): δ5.27-5.44(m,12H),3.48-3.79(m,3H),2.63-3.00(m,12H),2. 16-2.61(m,12H),2.05(q,J=6.80Hz,12H),1.18-1.57(m,51H),0.89(t,J=6.88Hz,9H)

[0199] ESI-MS: m / z 910.8 [M+H] + 911.8[M+2H] + 912.8 [M+3H] +

[0200] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. SEQUENCE LISTING <110> Beijing Qichen Biotechnology Co., Ltd. <120> S protein mutant of novel coronavirus strain and its genetically engineered mRNA and vaccine composition <130> CPCN22410423 <160> 9 <170> PatentIn version 3.5 <210> 1 <211> 1273 <212> PRT <213> Unknown <220> <223> 2019-nCoV wild-type S protein <400> 1 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala 675 680 685 Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser 690 695 700 Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile 705 710 715 720 Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val 725 730 735 Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu 740 745 750 Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr 755 760 765 Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln 770 775 780 Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe 785 790 795 800 Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser 805 810 815 Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly 820 825 830 Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp 835 840 845 Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu 850 855 860 Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly 865 870 875 880 Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile 885 890 895 Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr 900 905 910 Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn 915 920 925 Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala 930 935 940 Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn 945 950 955 960 Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val 965 970 975 Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln 980 985 990 Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val 995 1000 1005 Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn 1010 1015 1020 Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys 1025 1030 1035 Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 1040 1045 1050 Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val 1055 1060 1065 Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His 1070 1075 1080 Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn 1085 1090 1095 Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln 1100 1105 1110 Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val 1115 1120 1125 Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro 1130 1135 1140 Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn 1145 1150 1155 His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn 1160 1165 1170 Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu 1175 1180 1185 Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu 1190 1195 1200 Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu 1205 1210 1215 Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met 1220 1225 1230 Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys 1235 1240 1245 Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro 1250 1255 1260 Val Leu Lys Gly Val Lys Leu His Tyr Thr 1265 1270 <210> 2 <211> 1206 <212> PRT <213> Unknown <220> <223> 2019-nCoV S protein mutant <400> 2 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Arg Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Ile Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Asp Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Val Tyr Ser Ser 145 150 155 160 Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp 165 170 175 Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe 180 185 190 Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile 195 200 205 Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu 210 215 220 Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu 225 230 235 240 Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp 245 250 255 Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr 260 265 270 Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp 275 280 285 Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe 290 295 300 Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro 305 310 315 320 Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe 325 330 335 Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn 340 345 350 Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn 355 360 365 Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys 370 375 380 Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile 385 390 395 400 Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile 405 410 415 Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile 420 425 430 Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn 435 440 445 Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg 450 455 460 Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys Pro Cys Asn Gly 465 470 475 480 Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln 485 490 495 Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser 500 505 510 Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser 515 520 525 Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu 530 535 540 Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe 545 550 555 560 Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp 565 570 575 Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly 580 585 590 Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val 595 600 605 Leu Tyr Gln Gly Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala 610 615 620 Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser Asn Val 625 630 635 640 Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val Asn Asn 645 650 655 Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr 660 665 670 Gln Thr Gln Thr Asn Ser Arg Gly Ser Ala Ser Ser Val Ala Ser Gln 675 680 685 Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala 690 695 700 Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val 705 710 715 720 Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys 725 730 735 Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu 740 745 750 Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile 755 760 765 Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys 770 775 780 Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe 785 790 795 800 Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Pro Ile 805 810 815 Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile 820 825 830 Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile 835 840 845 Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr 850 855 860 Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile 865 870 875 880 Thr Ser Gly Trp Thr Phe Gly Ala Gly Pro Ala Leu Gln Ile Pro Phe 885 890 895 Pro Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn 900 905 910 Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala 915 920 925 Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Pro Ser Ala Leu Gly 930 935 940 Lys Leu Gln Asn Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu 945 950 955 960 Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn 965 970 975 Asp Ile Leu Ser Arg Leu Asp Pro Pro Glu Ala Glu Val Gln Ile Asp 980 985 990 Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln 995 1000 1005 Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala 1010 1015 1020 Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val 1025 1030 1035 Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser 1040 1045 1050 Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala 1055 1060 1065 Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly 1070 1075 1080 Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr 1085 1090 1095 His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile 1100 1105 1110 Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile 1115 1120 1125 Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu 1130 1135 1140 Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr 1145 1150 1155 Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser 1160 1165 1170 Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala 1175 1180 1185 Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys 1190 1195 1200 Tyr Glu Gln 1205 <210> 3 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Domain for assisting in trimer formation <400> 3 Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys 1 5 10 15 Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly 20 25 <210> 4 <211> 3618 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 4 atgttcgtgt tcctcgtgct ccttccgctg gtctcgagcc agtgcgtcaa tttgcgcacg 60 aggacgcagt tgccccccgc gtacacgaac tcgtttacgc ggggggtgta ctacccggac 120 aaggtcttcc gcagctctgt cctgcacagc actcaggacc tcttcctccc gttcttctcg 180 aacgtgacgt ggttccacgc cattcacgtg tcggggacga acgggacgaa gaggttcgac 240 aaccctgttc tgccgttcaa cgacggggtg tacttcgctt cgatcgagaa gtccaacatt 300 attcgcgggt ggatattcgg gaccactctc gattcgaaga ctcagtcctt gctgatagtg 360 aacaacgcca cgaacgtggt cattaaggtc tgcgagttcc agttctgtaa tgacccgttc 420 ctggacgttt actatcacaa gaacaacaag tcttggatgg agagtgaggt gtattcgtcc 480 gcgaataatt gtaccttcga gtatgtctcg cagccattct tgatggatct tgagggcaag 540 cagggaaatt tcaagaatct ccgcgagttt gtcttcaaga acatcgacgg gtacttcaag 600 atatactcga agcacacgcc gatcaacctc gtccgtgatc tcccgcaggg cttcagcgct 660 ctggagccgc tggtggatct cccgatcggg atcaacatca cgcggttcca gacgctgctg 720 gccctgcaca ggagttacct gacgccgggt gactccagta gtgggtggac tgcgggtgcc 780 gcggcgtact acgtcgggta cctgcagccg cgcacgttct tgttgaagta caacgagaac 840 gggacgatca cggacgcggt tgattgcgcg ttggaccctc tgtcggagac gaagtgcacc 900 ctgaagtcgt tcacggtgga gaagggtatc tatcagacct cgaacttccg ggtccagccg 960 actgagagta tcgttcggtt cccgaacatt acgaacctgt gtccgttcgg ggaggtcttc 1020 aacgcgacgc ggttcgcgag tgtgtacgct tggaaccgga agaggatctc gaattgtgtg 1080 gcggactaca gtgtgctgta caattcggcg tccttttcca cgttcaagtg ctacggggtg 1140 tcgcccacga agttgaacga cctctgcttc accaacgtgt atgcggattc cttcgtcatc 1200 cgtggtgacg aggtgcgtca gattgcgccg gggcagacgg ggaagatagc ggactataat 1260 1320 agtaaggtcg ggggcaacta taattatcgg taccgtctgt tccggagaag caatctgaag 1380 cccttgagc gcgatatctc gaccgagatc taccaggccg gctcgaagcc gtgcaacggc 1440 gtcgaggggt ttaattgtta ctttccgtta cagagctacg ggtttcagcc cacgaacggg 1500 gtggggtacc agccctaccg cgtcgtggtg ctgagcttcg agctgctgca cgccccggcc 1560 1620 1680 cagcagttcg gtcgggatat cgcggacacc acggatgccg tgagggatcc gcagacgctt 1740 gagattctgg acatcacgcc ctgcagcttc gggggcgtca gtgtgatcac gcctggtacg 1800 aacaccagca accaggttgc ggtgttgtac cagggtgtga attgcactga ggtccccgta 1860 gcgatccacg cggatcagct gaccccgacg tggagggtgt actcgacggg gagtaatgtc 1920 ttccagactc gcgcgggttg cctgattggc gctgagcacg tgaacaactc gtacgagtgc 1980 gacattccca ttggggcggg gatctgcgcg tcgtaccaga cccagacgaa cagccggggc 2040 agcgctagca gcgtcgcgtc gcagtcgatc atcgcgtaca cgatgagcct gggggcggag 2100 aacagtgtgg cctattcgaa caacagcata gctatcccca cgaattttac gatcagtgtg 2160 acgaccgaga tcttgcccgt gtcgatgacc aagacctcgg tcgattgcac gatgtacatt 2220 tgtggggata gcactgagtg ttctaacctc ctgctccagt acggcagttt ctgtacgcag 2280 ctcaaccggg cgcttacggg gattgccgtg gagcaggaca agaacactca ggaggtgttt 2340 gcgcaggtca agcagatcta caagacgcct ccgatcaagg atttcggggg gttcaatttc 2400 tcccagatac tccccgaccc ttcgaagccc agcaagcgta gccctattga ggacctgctc 2460 ttcaataagg ttacgcttgc ggacgcgggc ttcatcaagc agtacgggga ctgtctgggg 2520 gacattgccg cccgggacct gatctgtgct cagaagttca atgggctcac tgttctgccg 2580 cccctgctca cggacgagat gatcgcgcag tacacgtcgg cgctcctcgc cggcacgatc 2640 acgtcgggct ggacgtttgg ggctggtcct gcgctgcaga tcccgttccc tatgcagatg 2700 gcgtaccgct tcaatgggat cggggtgacc cagaatgtcc tgtacgagaa tcagaagctc 2760 atcgccaatc agttcaactc ggcgatcggg aagatacagg actccctgtc gagtacgcct 2820 tccgcgttgg ggaagctgca gaacgtggtg aaccagaatg ctcaggcgtt gaacacgttg 2880 gtgaagcagc tgtcgtccaa cttcggggcg atatcctcgg tgctgaacga tattctcagt 2940 cggctggacc cgccggaggc ggaggttcag atcgatagac tcatcactgg tcgctctccag 3000 agtttgcaga cgtacgtgac tcagcagctc atccgggctg ctgagatacg tgcgtctgcg 3060 aacctggcgg cgaccaagat gagtgagtgc gtgctggggc agagcaagcg ggtggacttt 3120 tgcgggaagg gctatcacct gatgtccttc ccgcagtccg cccctcacgg ggtggtcttc 3180 ctgcacgtga cgtatgtgcc ggcgcaggag aagaacttca ccacggcgcc ggccatatgt 3240 cacgacggga aggcccactt cccccgtgag ggggtcttcg tgtcgaatgg gacgcactgg 3300 ttcgtgacgc agcggaattt ctatgagccg cagataatta cgactgacaa cacgtttgtc 3360 agtggtaatt gtgatgtggt catagggatt gttaacaaca ccgtgtatga tcccctccag 3420 ccggagctgg acagcttcaa ggaggagctg gataagtact tcaagaatca cacgtcgccg 3480 gacgtggatc ttggggacat atcggggatc aacgcgagtg ttgttaacat acagaaggag 3540 atcgaccggc tcaatgaggt tgcgaagaac ctcaatgagt cgttgatcga ccttcaggag 3600 ctcggcaagt atgagcag 3618 <210> 5 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <~400> 5 ggctatatcc cagaggcccc tagagatggc caggcctacg ttagaaagga cggcgagtgg 60 gtcctgctga gcacattcct gggc 84 <210> 6 <211> 50 <212> RNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 6 acauuugcuu cugacacaac uguguucacu agcaaccuca aacagacacc 50 <210> 7 <211> 88 Note: In the translation, for the Chinese part "<223> 人工序列", it is translated as "<223> Artificial Sequence" as per the requirement. Also, "<~400>" is translated as "<400>" as it seems to be a misprint in the original and the pattern suggests it should be the same as the other "<400>" tags. And for the RNA sequence part, it is left in the original format as it contains non-standard nucleotide abbreviations which are likely specific to a certain context and not meant to be translated in a traditional sense. If there is more context available for the RNA sequence, a more accurate translation might be possible. <212> RNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 7 gcuggagccu cgguagccgu uccuccugcc cgcugggccu cccaacgggc ccuccucccc 60 uccuugcacc ggcccuuccu ggucuuug 88 <210> 8 <211> 3705 <212> RNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 8 auguucgugu uccucgugcu ccuuccgcug gucucgagcc agugcgucaa uuugcgcacg 60 aggacgcagu ugccccccgc guacacgaac ucguuuacgc ggggggugua cuacccggac 120 aaggucuucc gcagcucugu ccugcacagc acucaggacc ucuuccuccc guucuucucg 180 aacgugacgu gguuccacgc cauucacgug ucggggacga acgggacgaa gagguucgac 240 aacccuguuc ugccguucaa cgacggggug uacuucgcuu cgaucgagaa guccaacauu 300 auucgcgggu ggauauucgg gaccacucuc gauucgaaga cucaguccuu gcugauagug 360 aacaacgcca cgaacguggu cauuaagguc ugcgaguucc aguucuguaa ugacccguuc 420 cuggacguuu acuaucacaa gaacacaag ucuuggaugg agagugaggu guauucgucc 480 gcgaauaauu guaccuucga guaugucucg cagccaucu ugauggaucu ugaggggcaag 540 cagggaauu ucaagaaucu ccgcgaguuu gucuucaaga acucgacgg guacuucaag 600 auauacucga agcacacgcc gaucaaccuc guccgugauc ucccgcagggg cucagcgcu 660 cuggagccgc ugguggaucu cccgaucggg aucacauca cgcgguucca gacgcugcug 720 gcccugcaca ggaguuaccu gacgccgggu gacuccagua guggguggac ugcgggugcc 780 gcggcguacu acgucgggua ccugcagccg cgcacguacu ugugaagua cacgagaac 840 gggacgauca cggacgcggu ugauugcgcg uuggacccuc ugucggac gaagugcacc 900 cugaagucgu ucacggugga gaaggguauc uaucagaccu cgaacuuccg gguccagccg 960 acugagagua ucguucgguu cccgaacaou acgaaccugu guccguucgg ggaggucuuc 1020 aacgcgacgc gguucgcgag uguacgcu uggaccgga agggcuc gauugugug 1080 gcggacuaca gugugcugua caauucggcg uccuuuucca cguucaagug cuacggggg 1140 ucgcccacga aguugaacga ccucugcuuc accaacgugu augcggauuc cuucgucauc 1200 cguggugacg aggugcguca gauugcgccg gggcagacgg ggaagauagc ggacuauaau 1260 uauaaguugc ccgacgacuu uacuggcugc guuauugcuu ggaacagcaa uaaccuggac 1320 aguaaggucg ggggcaacua uaauuaucgg uaccgucugu uccggaagag caaucugaag 1380 cccuucgagc gcgauaucuc gaccgagauc uaccaggccg gcucgaagcc gugcaacggc 1440 gucgaggggu uuaauuguua cuuuccguua cagagcuacg gguuucagcc cacgaacggg 1500 gugggguacc agcccuaccg cgucguggug cugagcuucg agcugcugca cgccccggcc 1560 acggugugcg guccgaagaa aaguacaaac cuugugaaga acaagugugu gaacuuuaac 1620 uucaacgggc ucaccgggac ggggguguug acggagagua acaagaaguu ccugccguuc 1680 cagcaguucg gucgggauau cgcggacacc acggaugccg ugagggaucc gcagacgcuu 1740 gagauucugg acaucacgcc cugcagcuuc gggggcguca gugugaucac gccugguacg 1800 aacaccagca accagguugc gguguuguac caggguguga auugcacuga gguccccgua 1860 1920 1980 gacauuccca uugggggcggg gaucugcgcg ucguaccaga cccagacgaa cagccggggc 2040 agcgcuagca gcgucgcguc gcagucgauc aucgcguaca cgaugagccu gggggcggag 2100 aacagugugg ccuauucgaa caacagcaua gcuaucccca cgaauuuuac gaucagugug 2160 2220 ugggggaua gacugagug uucuaaccuc cugcuccagu acggcaguuu cuguacgcag 2280 cucaaccggg cgcuuacggg gauugccgug gagcaggaca agaacacua ggagguguuu 2340 gcgcagguca agcagaucua caagacgccu ccgaucaagg auuucggggg guucaauuuc 2400 ucccagauac uccccgaccc uucgaagccc agcaagcgua gcccuauuga ggaccugcuc 2460 uucaauaagg uaacgcuugc ggacgcgggc uucaucaagc aguacggga cugucugggg 2520 gacauugccg cccgggaccu gaucugugcu cagaaguuca augggcucac uguucugccg 2580 ccccugcuca cggacgagau gaucgcgcag uacacgucgg cgcuccucgc cggcacgauc 2640 acgucgggcu ggacguuugg ggcugguccu gcgcugcaga ucccguuccc uaugcagaug 2700 gcguaccgcu ucaaugggau cggggugacc cagaaugucc ugaacgagaa ucagaagcuc 2760 aucgccaauc aguacaacuc ggcgaucgggg aagauacagg acucccuguc gaguacgccu 2820 uccgcguugg ggaagcugca gaacguggug aaccagaaug cucaggcguu gaacacguug 2880 gugaagcagc ugucguccaa cuucggggcg auauccucgg ugcugaacga uauucucagu 2940 cggcuggacc cgccggaggc ggagguucag aucgauagac ucaucacugg ucgccuccag 3000 3060 aaccuggcgg cgaccaagau gagugagugc gugcuggggc agagcaagcg gguggacuuu 3120 ugcgggaagg gcuaucaccu gauguccuuc ccgcaguccg ccccucacgg gggugucuuc 3180 cugcacguga cguaugugcc ggcgcaggag aagaacuuca ccacggcgcc ggccauaugu 3240 cacgacggga aggcccacuu cccccgugag ggggucuucg ugucgaaugg gacgcacugg 3300 uucgugacgc agcggaauuu cuaugagccg cagauaauua cgacugacaa cacguuuguc 3360 agugguaauu gugauguggu cauagggauu guuaacaaca ccguguauga uccccuccag 3420 ccggagcugg acagcuucaa ggaggagcug gauaaguacu ucaagaauca cacgucgccg 3480 gacguggauc uuggggacau aucggggauc aacgcgagug uuguuaacau acagaaggag 3540 aucgaccggc ucaaugaggu ugcgaagaac cucaaugagu cguugaucga ccuucaggag 3600 cucggcaagu augagcaggg cuauauccca gaggccccua gagauggcca ggccuacguu 3660 agaaaggacg gcgagugggu ccugcugagc acauuccugg gcuga 3705 <210> 9 <211> 4098 <212> RNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 9 gggagaccgg ccucgagaca uuugcuucug acacaacugu guucacuagc aaccucaaac 60 agacaccaag cuugccacca uguucguguu ccucgugcuc cuuccgcugg ucucgagcca 120 gugcgucaau uugcgcacga ggacgcaguu gccccccgcg uacacgaacu cguuuacgcg 180 ggggguguac uacccggaca aggucuuccg cagcucuguc cugcacagca cucaggaccu 240 cuuccucccg uucuucucga acgugacgug guuccacgcc auucacgugu cggggacgaa 300 cgggacgaag agguucgaca acccuguucu gccguucaac gacggggugu acuucgcuuc 360 gaucgagaag uccaacauua uucgcgggug gauauucggg accacucucg auucgaagac 420 ucaguccuug cugauaguga acaacgccac gaacgugguc auuaaggucu gcgaguucca 480 guucuguaau gacccguucc uggacguuua cuaucacaag aacaacaagu cuuggaugga 540 gagugaggug uauucguccg cgaauaauug uaccuucgag uaugucucgc agccauucuu 600 gauggaucuu gagggcaagc agggaaauuu caagaaucuc cgcgaguuug ucuucaagaa 660 caucgacggg uacuucaaga uauacucgaa gcacacgccg aucaaccucg uccgugaucu 720 cccgcagggc uucagcgcuc uggagccgcu gguggaucuc ccgaucggga ucaacaucac 780 gcgguuccag acgcugcugg cccugcacag gaguuaccug acgccgggug acuccaguag 840 uggguggacu gcgggugccg cggcguacua cgucggguac cugcagccgc gcacguucuu 900 guugaaguac aacgagaacg ggacgaucac ggacgcgguu gauugcgcgu uggacccucu 960 gucggagacg aagugcaccc ugaagucguu cacgguggag aaggguaucu aucagaccuc 1020 gaacuuccgg guccagccga cugagaguau cguucgguuc ccgaacauua cgaaccugug 1080 uccguucggg gaggucuuca acgcgacgcg guucgcgagu guguacgcuu ggaaccggaa 1140 gaggaucucg aauugugugg cggacuacag ugugcuguac aauucggcgu ccuuuuccac 1200 guucaagugc uacggggugu cgcccacgaa guugaacgac cucugcuuca ccaacgugua 1260 ugcggauucc uucgucaucc guggugacga ggugcgucag auugcgccgg ggcagacggg 1320 gaagauagcg gacuauaauu auaaguugcc cgacgacuuu acuggcugcg uuauugcuug 1380 gaacagcaau aaccuggaca guaaggucgg gggcaacuau aauuaucggu accgucuguu 1440 ccggagaagc aaucugaagc ccuucgagcg cgauauuccg accgagaucu accaggccgg 1500 cucgaagccg ugcaacggcg ucgagggguu uaauuguuac uuuccguuac agagcuacgg 1560 guuucagccc acgaacgggg ugggguacca gcccuaccgc gucguggugc ugagcuucga 1620 gcugcugcac gccccggcca cggugugcgg uccgaagaaa aguacaaacc uugugaagaa 1680 caagugugug aacuuuaacu ucaacgggcu caccgggacg gggguguuga cggagaguaa 1740 caagaaguuc cugccguucc agcaguucgg ucgggauauc gcggacacca cggaugccgu 1800 gagggauccg cagacgcuug agauucugga caucacgccc ugcagcuucg ggggcgucag 1860 ugugaucacg ccugguacga acaccagcaa ccagguugcg guguuguacc agggugugaa 1920 uugcacugag guccccguag cgauccacgc ggaucagcug accccgacgu ggagggugua 1980 cucgacgggg aguaaugucu uccagacucg cgcggguugc cugauuggcg cugagcacgu 2040 gaacaacucg uacgagugcg acauucccau uggggcgggg aucugcgcgu cguaccagac 2100 ccagacgaac agccggggca gcgcuagcag cgucgcgucg cagucgauca ucgcguacac 2160 gaugagccug ggggcggaga acaguguggc cuauucgaac aacagcauag cuauccccac 2220 gaauuuuacg aucaguguga cgaccgagau cuugcccgug ucgaugacca agaccucggu 2280 cgauugcacg auguacauuu guggggauag cacugagugu ucuaaccucc ugcuccagua 2340 cggcaguuuc ugaacgcagc ucaaccgggc gcuuacgggg augccgugg agcaggacaa 2400 gaacacucag gaggguguuug cgcaggucaa cgagaucuac aagacgccuc cgaucaagga 2460 uuucgggggg uucaauuucu cccagauacu ccccgacccu ucgaagccca gcaagcguag 2520 cccuauugag gaccugcucu ucaauaaggu uacgcuugcg gacgcgggcu ucaucaagca 2580 guacggggac ugucuggggg acauugccgc ccgggaccug aucugugcuc agaaguucaa 2640 ugggcucacu guucugccgc cccugcucac ggacgagaug aucgcgcagu acacgucggc 2700 gcuccucgcc ggcacgauca cgucgggcug gacguuuggg gcugguccug cgcugcagau 2760 cccguucccu augcagaugg cguaccgcuu caaugggauc ggggugaccc agaauguccu 2820 2880 cucccugucg aguacgccuu ccgcguuggg gaagcugcag aacgugguga accagaaugc 2940 ucaggcguug aacacguugg ugaagcagcu gucguccaac uucggggcga uauccucggu 3000 gcugaacgau auucucaguc ggcuggacccc gccggaggcg gagguucaga ucgauagacu 3060 caucacugu cgccuccaga guugcagac guacgugacu cagcagcuca uccgggcugc 3120 ugagauacgu gcgucugcga accuggcggc gaccaagaug agugagugcg ugcuggggca 3180 gagcaagcgg guggacuuuu gcgggaaggg cuaucaccug auguccuucc cgcaguccgc 3240 cccucacggg guggucuucc ugcacgugac guaugugccg gcgcaggaga agacuucac 3300 cacggcgccg gccauauguc acgacgggaa ggcccacuuc ccccgugagg gggucuucgu 3360 gucgaauggg acgcacuggu ucgugacgca gcggaauuuc uaugagccgc aguaauuac 3420 gacugacaac acguuuguca gugguaauug ugaugugguc auagggauug uuaacaacac 3480 cguguaugau ccccuccagc cggagcugga cagcuaag gaggagcugg auaaguacuu 3540 caagaaucac acgucgccgg acguggaucu uggggacaua ucgggggauca acgcgagugu 3600 uguuaacaua cagaaggaga ucgaccggcu caaugagguu gcgaagaacc ucaaugaguc 3660 guugaucgac cuucaggagc ucggcaagua ugagcagggc uauaucccag aggccccuag 3720 agauggccag gccuacguua gaaggacgg cgaguggguc cugcugagca cauuccuggg 3780 cugagaauuc gcuggagccu cgguagccgu uccuccugcc cgcugggccu cccaacgggc 3840 ccuccucccc uccuugcacc ggcccuuccu ggucuuuggc uggagccucg guagccguuc 3900 cuccugcccg cugggccucc caacgggccc uccuccccuc cuugcaccgg cccuuccugg 3960 ucuuuguuaa uuaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4020 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4080 aaaaaaaaaa aaaacuag 4098

Claims

1. A mutant of the S protein of 2019-nCoV, characterized in that, Based on the amino acid sequence shown in SEQ ID NO:1, its extracellular domain has the following amino acid mutations relative to the extracellular domain of the parental S protein: F817P, A892P, A899P, A942P and KV986_987PP, as well as T19R, G142D, EF156_157del, R158G, L452R, T478K, D614G, P681R, D950N, and amino acids 682-685 RRAR mutation to GSAS; The 2019-nCoV S protein mutant does not contain the transmembrane domain and cytoplasmic tail of the S protein; The 2019-nCoV S protein mutant directly fuses the auxiliary trimeric domain to the C-terminus of the extracellular domain.

2. The 2019-nCoV S protein mutant as described in claim 1, characterized in that, It contains an amino acid sequence as shown in SEQ ID NO:

2.

3. The 2019-nCoV S protein mutant as described in claim 1 or 2, characterized in that, The structural domain that assists in the formation of the trimer is the T4 Fibritin Foldon Trimerization Motif.

4. The 2019-nCoV S protein mutant as described in claim 3, characterized in that, The S protein mutant of 2019-nCoV has an amino acid sequence that is a direct link from the N-terminus to the C-terminus, as shown in SEQ ID NO:2 and SEQ ID NO:

3.

5. A DNA molecule, characterized in that, The DNA molecule encodes the S protein mutant of 2019-nCoV as described in any one of claims 1-4.

6. The DNA molecule as described in claim 5, characterized in that, The nucleotide sequence of the DNA molecule consists of the nucleotide sequences shown in SEQ ID NO:4 and SEQ ID NO:5, which are directly linked from the 5' end to the 3' end.

7. An expression carrier, characterized in that, It comprises the DNA molecule as described in claim 5 or 6.

8. A cell, characterized in that, It comprises the DNA molecule of claim 5 or 6 or the expression vector of claim 7.

9. An mRNA molecule, characterized in that, An open reading frame comprising an S protein mutant of 2019-nCoV as described in any one of claims 1-4.

10. The mRNA molecule as described in claim 9, characterized in that, The nucleotide sequence of the open reading frame is shown in SEQ ID NO:

8.

11. The mRNA molecule as described in claim 9 or 10, characterized in that, The mRNA contains a 5' UTR, an open reading frame encoding the S protein mutant of 2019-nCoV, a 3' UTR, and a poly-A tail from the 5' end to the 3' end.

12. The mRNA molecule as claimed in claim 11, characterized in that, The nucleotide sequence of the 5'UTR is shown in SEQ ID NO:

6.

13. The mRNA molecule as described in claim 11, characterized in that, The nucleotide sequence of the 3'UTR consists of two nucleotide sequences shown in SEQ ID NO:7, linked end-to-end.

14. The mRNA molecule as claimed in claim 11, characterized in that, The poly-A tail is 50-200 nucleotides in length.

15. The mRNA molecule as described in claim 14, characterized in that, The poly-A tail is 100-150 nucleotides in length.

16. The mRNA molecule as claimed in claim 11, characterized in that, The mRNA further contains a Kozak sequence, which is GCCACC.

17. The mRNA molecule as claimed in claim 11, characterized in that, The mRNA further includes a 5' cap, which is CAP1.

18. The mRNA molecule as described in claim 9, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

9.

19. A nucleic acid molecule, characterized in that, It encodes the mRNA molecule as described in any one of claims 9-18.

20. A vaccine composition, characterized in that, It comprises the S protein mutant of 2019-nCoV as described in any one of claims 1-4, or the mRNA molecule as described in any one of claims 9-18.

21. The vaccine composition according to claim 20, characterized in that, The vaccine composition also contains pharmaceutically acceptable excipients and / or adjuvants.

22. The vaccine composition of claim 20, further comprising lipid nanoparticles, wherein mRNA is located within the lipid nanoparticles, and the lipid nanoparticles contain 30-60 mol% of ionizable / cationic lipid molecules, 5-30 mol% of neutral lipid molecules, 30-50 mol% of cholesterol lipid molecules, and 0.4-10 mol% of PEGylated lipid molecules, comprising a total lipid molecule content of 30-60 mol% of ionizable / cationic lipid molecules, 5-30 mol% of neutral lipid molecules, 30-50 mol% of cholesterol lipid molecules, and 0.4-10 mol% of PEGylated lipid molecules.

23. The vaccine composition according to claim 22, characterized in that, The lipid nanoparticles contain 32-55 mol% ionizable / cationic lipid molecules, 8-20 mol% neutral lipid molecules, 35-50 mol% cholesterol lipid molecules, and 0.5-5 mol% PEGylated lipid molecules.

24. The vaccine composition according to claim 22, characterized in that, The lipid nanoparticles contain 39-51 mol% ionizable / cationic lipid molecules, 9-16 mol% neutral lipid molecules, 37-49 mol% cholesterol lipid molecules, and 1.3-2.7 mol% PEGylated lipid molecules.

25. The vaccine composition according to any one of claims 22-24, characterized in that, The ionizable / cationic lipid molecule is a compound represented by formula C, formula C Each n3 is independent of each other, either the same or different, and each n3 is selected from integers from 1 to 8. Each m3 is independent of each other, either the same or different, and each m3 is selected from integers from 0 to 8.

26. The vaccine composition according to claim 25, characterized in that, Each n3 is selected from integers from 4 to 8, and each m3 is selected from integers from 4 to 8.

27. The vaccine composition according to claim 25, characterized in that, Each n3 is identical to the others, and each m3 is identical to the others.

28. The vaccine composition according to claim 25, characterized in that, The ionizable / cationic lipid molecule has the following structure: 。 29. The vaccine composition according to any one of claims 22-24, characterized in that, The total mass ratio of lipid molecules to mRNA is 5-20:1.

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